Transgenic plants expressing photorhabdus toxin

ABSTRACT

Novel polynucleotide sequences that encode insect toxins TcdA and TcbA have base compositions that differ substantially from the native genes, making them more similar to plant genes. The new sequences are suitable for use for high expression in both monocots and dicots. Transgenic plants with a genome comprising the nucleic acid of SEQ ID NO:4 are insect resistant.

RELATED APPLICATION

This application is a divisional application of application Ser. No. 09/637,048, filed Aug. 11, 2000, now U.S. Pat. No. 6,590,142, which claims benefit of U.S. Provisional Patent Application No. 60/148,356, filed Aug. 11, 1999.

BACKGROUND OF THE INVENTION

As reported in WO98/08932, protein toxins from the genus Photorhabdus have been shown to have oral toxicity against insects. The toxin complex produced by Photorhabdus luminescens (W-14), for example, has been shown to contain ten to fourteen proteins, and it is known that these are produced by expression of genes from four distinct genomic regions: tca, tcb, tcc, and tcd. WO98/08932 discloses nucleotide sequences for the native toxin genes.

Of the separate toxins isolated from Photorhabdus luminescens (W-14), those designated Toxin A and Toxin B are especially potent against target insect species of interest, for example corn rootworm. Toxin A is comprised of two different subunits. The native gene tcdA (SEQ ID NO:1) encodes protoxin TcdA (see SEQ ID NO:1). As determined by mass spectrometry, TcdA is processed by one or more proteases to provide Toxin A. More specifically, TcdA is an approximately 282.9 kDA protein (2516 aa) that is processed to provide TcdAii, an approximately 208.2 kDA (1849 aa) protein encoded by nucleotides 265-5811 of SEQ ID NO:1, and TcdAiii, an approximately 63.5 kDA (579 aa) protein encoded by nucleotides 5812-7551 of SEQ ID NO:1.

Toxin B is similarly comprised of two different subunits. The native gene tcbA (SEQ ID NO:2) encodes protoxin TcbA (see SEQ ID NO:2). As determined by mass spectrometry, TcbA is processed by one or more proteases to provide Toxin B. More specifically, TcbA is an approximately 280.6 kDA (2504 aa) protein that is processed to provide TcbAii, an approximately 207.7 kDA (1844 aa) protein encoded by nucleotides 262-5793 of SEQ ID NO:2 and TcbAiii, an approximately 62.9 kDA (573 aa) protein encoded by nucleotides 5794-7512 of SEQ ID NO:2.

The native tcdA and tcbA genes are not well suited for high level expression in plants. They encode multiple destabilization sequences, mRNA splice sites, polyA addition sites and other possibly detrimental sequence motifs. In addition, the codon compositions are not like those of plant genes. WO98/08932 gives general guidance on how the toxin genes could be reengineered to more efficiently expressed in the cytoplasm of plants, and describes how plants can be transformed to incorporate the Photorhabdus toxin genes into their genomes.

SUMMARY OF THE INVENTION

In a preferred embodiment, the invention provides novel polynucleotide sequences that encode TcdA and TcbA. The novel sequences have base compositions that differ substantially from the native genes, making them more similar to plant genes. The new sequences are suitable for use for high expression in both monocots and dicots, and this feature is designated by referring to the sequences as the “hemicot” criteria, which is set forth in detail hereinafter. Other important features of the sequences are that potentially deleterious sequences have been eliminated, and unique restriction sites have been built in to enable adding or changing expression elements, organellar targeting signals, engineered protease sites and the like, if desired.

In a particularly preferred embodiment, the invention provides polynucleotide sequences that satisfy hemicot criteria and that comprise a sequence encoding an endoplasmic reticulum signal or similar targeting sequence for a cellular organelle in combination with a sequence encoding TcdA or TdbA.

More broadly, the invention provides engineered nucleic acids encoding functional Photorhabdus toxins wherein the sequences satisfy hemicot criteria.

The invention also provides transgenic plants with genomes comprising a novel sequence of the invention that imparts functional activity against insects.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO:1 is the native tcdA DNA sequence together with the corresponding encoded amino acid sequence for TcdA.

SEQ ID NO:2 is the native tcbA DNA sequence together with the corresponding encoded amino acid sequence for TcbA.

SEQ ID NO:3 is an artificial sequence encoding TcdA that is suitable for expression in monocot and dicot plants.

SEQ ID NO:4 is an artificial sequence encoding TcbA that is suitable for expression in monocot and dicot plants.

SEQ ID NO:5 is an artificial hemicot sequence that encodes the 21 amino acid ER signal peptide of 15 kDa zein from Black Mexican Sweet maize.

SEQ ID NO:6 is an artificial hemicot sequence that encodes for the full-length native TcdA protein (amino acids 22-2537) fused to the modified 15 kDa zein endoplasmic reticulum signal peptide (amino acids 1-21).

DETAILED DESCRIPTION

The native Photorhabdus toxins are protein complexes that are produced and secreted by growing bacteria cells of the genus Photorhabdus. Of particular interest are the proteins produced by the species Photorhabdus luminescens. The protein complexes have a molecular size of approximately 1,000 kDa and can be separated by SDS-PAGE gel analysis into numerous component proteins. The toxins contain no hemolysin, lipase, type C phospholipase, or nuclease activities. The toxins exhibit significant toxicity upon ingestion by a number of insects.

A unique feature of Photorhabdus is its bioluminescence. Photorhabdus may be isolated from a variety of sources. One such source is nematodes, more particularly nematodes of the genus Heterorhabditis. Another such source is from human clinical samples from wounds, see Farmer et al. 1989 J. Clin. Microbiol. 27 pp. 1594-1600. These saprohytic strains are deposited in the American Type Culture Collection (Rockville, Md.) ATCC #s 43948, 43949, 43950, 43951, and 43952, and are incorporated herein by reference. It is possible that other sources could harbor Photorhabdus bacteria that produce insecticidal toxins. Such sources in the environment could be either terrestrial or aquatic based.

The genus Photorhabdus is taxonomically defined as a member of the Family Enterobacteriaceae, although it has certain traits a typical of this family. For example, strains of this genus are nitrate reduction negative, yellow and red pigment producing and bioluminescent. This latter trait is otherwise unknown within the Enterobacteriaceae. Photorhabdus has only recently been described as a genus separate from the Xenorhabdus (Boemare et al., 1993 Int. J. Syst. Bacteriol. 43, 249-255). This differentiation is based on DNA—DNA hybridization studies, phenotypic differences (e.g., presence (Photorhabdus) or absence (Xenorhabdus) of catalase and bioluminescence) and the Family of the nematode host (Xenorhabdus; Steinernematidae, Photorhabdus; Heterorhabditidae). Comparative, cellular fatty-acid analyses (Janse et al. 1990, Lett. Appl. Microbiol 10, 131-135; Suzuki et al. 1990, J. Gen. Appl. Microbiol., 36, 393-401) support the separation of Photorhabdus from Xenorhabdus.

Currently, the bacterial genus Photorhabdus is comprised of a single defined species, Photorhabdus luminescens (ATCC Type strain #29999, Poinar et al., 1977, Nematologica 23, 97-102). A variety of related strains have been described in the literature (e.g., Akhurst et al. 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare et al. 1993 Int. J. Syst. Bacteriol. 43 pp. 249-255; Putz et al. 1990, Appl. Environ. Microbiol., 56, 181-186).

The following toxin producing Photorhabdus strains have been deposited:

strain accession number date of deposit W-14 ATCC 55397 Mar. 5, 1993 WX1 NRRL B-21710 Apr. 29, 1997 WX2 NRRL B-21711 Apr. 29, 1997 WX3 NRRL B-21712 Apr. 29, 1997 WX4 NRRL B-21713 Apr. 29, 1997 WX5 NRRL B-21714 Apr. 29, 1997 WX6 NRRL B-21715 Apr. 29, 1997 WX7 NRRL B-21716 Apr. 29, 1997 WX8 NRRL B-21717 Apr. 29, 1997 WX9 NRRL B-21718 Apr. 29, 1997 WX10 NRRL B-21719 Apr. 29, 1997 WX11 NRRL B-21720 Apr. 29, 1997 WX12 NRRL B-21721 Apr. 29, 1997 WX14 NRRL B-21722 Apr. 29, 1997 WX15 NRRL B-21723 Apr. 29, 1997 H9 NRRL B-21727 Apr. 29, 1997 Hb NRRL B-21726 Apr. 29, 1997 Hm NRRL B-21725 Apr. 29, 1997 HP88 NRRL B-21724 Apr. 29, 1997 NC-1 NRRL B-21728 Apr. 29, 1997 W30 NRRL B-21729 Apr. 29, 1997 WIR NRRL B-21730 Apr. 29, 1997 B2 NRRL B-21731 Apr. 29, 1997 ATCC 43948 ATCC 55878 Nov. 5, 1996 ATCC 43949 ATCC 55879 Nov. 5, 1996 ATCC 43950 ATCC 55880 Nov. 5, 1996 ATCC 53951 ATCC 55881 Nov. 5, 1996 ATCC 43952 ATCC 55882 Nov. 5, 1996 DEPI NRRL B-21707 Apr. 29, 1997 DEP2 NRRL B-21708 Apr. 29, 1997 DEP3 NRRL B-21709 Apr. 29, 1997 P. zealandrica NRRL B-21683 Apr. 29, 1997 P. hepialus NRRL B-21684 Apr. 29, 1997 HB-Arg NRRL B-21685 Apr. 29, 1997 HB Oswego NRRL B-21686 Apr. 29, 1997 Hb Lewiston NRRL B-21687 Apr. 29, 1997 K-122 NRRL B-21688 Apr. 29, 1997 HMGD NRRL B-21689 Apr. 29, 1997 Indicus NRRL B-21690 Apr. 29, 1997 GD NRRL B-21691 Apr. 29, 1997 PWH-5 NRRL B-21692 Apr. 29, 1997 Megidis NRRL B-21693 Apr. 29, 1997 HF-85 NRRL B-21694 Apr. 29, 1997 A. Cows NRRL B-21695 Apr. 29, 1997 MP1 NRRL B-21696 Apr. 29, 1997 MP2 NRRL B-21697 Apr. 29, 1997 MP3 NRRL B-21698 Apr. 29, 1997 MP4 NRRL B-21699 Apr. 29, 1997 MP5 NRRL B-21700 Apr. 29, 1997 GL98 NRRL B-21701 Apr. 29, 1997 Gl101 NRRL B-21702 Apr. 29, 1997 GL138 NRRL B-21703 Apr. 29, 1997 GL155 NRRL B-21704 Apr. 29, 1997 GL217 NRRL B-21705 Apr. 29, 1997 GL257 NRRL B-21706 Apr. 29, 1997

All strains were deposited in accordance with the terms of the Budapest Treaty. Strains having accession numbers prefaced by “ATTC” were deposited on the indicated date in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA. Strains prefaced by “NRRL” were deposited on the indicated date in the Agricultural Research Service Patent Culture Collection (NRRL), National Center for Agricultural Utilization Research, ARS-USDA, 1815 North University St., Peoria Ill. 61604 USA.

The present invention provides hemicot nucleic acid sequences encoding toxins from any Photorhabdus species or strain that produces a toxin having functional activity. Hemicot nucleic acid sequences encoding proteins homologous to such toxins are also encompassed by the invention.

Several terms that are used herein have a particular meaning and are defined as follows:

By “functional activity” it is meant herein that the protein toxins) function as insect control agents in that the proteins are orally active, or have a toxic effect, or are able to disrupt or deter feeding, which may or may not cause death of the insect. When an insect comes into contact with an effective amount of toxin delivered via transgenic plant expression, formulated protein compositions), sprayable protein compositions), a bait matrix or other delivery system, the results are typically death of the insect, or the insects do not feed upon the source which makes the toxins available to the insects.

By “homolog” it is meant an amino acid sequence that is identified as possessing homology to a reference Photorhabdus toxin polypeptide amino acid sequence.

By “homology” it is meant an amino acid sequence that has a similarity index of at least 33% and/or an identity index of at least 26% to a reference Photorhabdus toxin polypeptide amino acid sequence, as scored by the GAP algorithm using the B10sum 62 protein scoring matrix Wisconsin Package Version 9.0, Genetics Computer Group GCG), Madison, Wis.).

By “identity” is meant an amino acid sequence that contains an identical residue at a given position, following alignment with a reference Photrhabdus toxin polypeptide amino acid sequence by the GAP algorithm.

By the use of the term “Photorhabdus toxin” it is meant any protein produced by a Photorhabdus microorganism strain which has functional activity against insects, where the Photorhabdus toxin could be formulated as a sprayable composition, expressed by a transgenic plant, formulated as a bait matrix, delivered via baculovirus, or delivered by any other applicable host or delivery system.

By the use of the term “toxic” or “toxicity” as used herein it is meant that the toxins produced by Photorhabdus have “functional activity” as defined herein.

By “substantial sequence homology” is meant either: a DNA fragment having a nucleotide sequence sufficiently similar to another DNA fragment to produce a protein having similar biochemical properties; or a polypeptide having an amino acid sequence sufficiently similar to another polypeptide to exhibit similar biochemical properties.

As with other bacterial toxins, the rate of mutation of the bacteria in a population causes many related toxins slightly different in sequence to exist. Toxins of interest here are those which produce protein complexes toxic to a variety of insects upon exposure, as described herein. Preferably, the toxins are active against Lepidoptera, Coleoptera, Homopotera, Diptera, Hymenoptera, Dictyoptera and Acarina. The inventions herein are intended to capture the protein toxins homologous to protein toxins produced by the strains herein and any derivative strains thereof, as well as any protein toxins produced by Photorhabdus. These homologous proteins may differ in sequence, but do not differ in function from those toxins described herein. Homologous toxins are meant to include protein complexes of between 300 kDa to 2,000 kDa and are comprised of at least two 2) subunits, where a subunit is a peptide which may or may not be the same as the other subunit. Various protein subunits have been identified and are taught in the Examples herein. Typically, the protein subunits are between about 18 kDa to about 230 kDa; between about 160 kDa to about 230 kDa; 100 kDa to 160 kDa; about 80 kDa to about 100 kDa; and about 50 kDa to about 80 kDa.

As discussed above, some Photorhabdus strains can be isolated from nematodes. Some nematodes, elongated cylindrical parasitic worms of the phylum Nematoda, have evolved an ability to exploit insect larvae as a favored growth environment. The insect larvae provide a source of food for growing nematodes and an environment in which to reproduce. One dramatic effect that follows invasion of larvae by certain nematodes is larval death. Larval death results from the presence of, in certain nematodes, bacteria that produce an insecticidal toxin which arrests larval growth and inhibits feeding activity.

Interestingly, it appears that each genus of insect parasitic nematode hosts a particular species of bacterium, uniquely adapted for symbiotic growth with that nematode. In the interim since this research was initiated, the name of the bacterial genus Xenorhabdus was reclassified into the Xenorhabdus and the Photorhabdus. Bacteria of the genus Photorhabdus are characterized as being symbionts of Heterorhabditus nematodes while Xenorhabdus species are symbionts of the Steinernema species. This change in nomenclature is reflected in this specification, but in no way should a change in nomenclature alter the scope of the inventions described herein.

The peptides and genes that are disclosed herein are named according to the guidelines recently published in the Journal of Bacteriology “Instructions to Authors” p. i-xii January 1996), which is incorporated herein by reference.

Transformation methods useful in carrying out the invention are well known, and are described, for example, in WO98/08932.

Hemicot tcdA and tcbA

SEQ ID NO: 3 is the nucleotide sequence for an engineered tcdA gene in accordance with the invention. SEQ ID NO: 4 is the nucleotide sequence for an engineered tcbA gene in accordance with the invention.

The following Tables 1 and 2 identify significant features of the engineered tcdA and tcbA genes.

TABLE 1 tcdA Feature nucleotides of SEQ ID NO:3 NcoI   1-6 HindIII  48-53 KpnI  246-254 sequence encoding  267-5798 TcbAii NheI  333-338 BglII 1215-1220 ClaI 2604-2609 PstI 4015-4020 AgeI 5088-5093 MunI 5598-5603 XbaI 5778-5783 sequence encoding 5799-7517 TcbAiii AflII 5853-5858 SphI 6439-6444 SfuI 7392-7397 SacI 7519-7524 XhoI 7522-7527 StuI 7528-7533 NotI 7533-7538

TABLE 2 tcbA Feature nucleotides of SEQ ID NO:5 NcoI   1-6 HindIII  48-53 KpnI  246-251 sequence encoding  267-5798 TcbAii NheI  333-338 BglII 1215-1220 ClaI 2604-2609 PstI 4015-4020 AgeI 5088-5093 MunI 5598-5603 XbaI 5778-5783 sequence 5799-7517 encoding TcbAiii AflII 5853-5858 SphI 6439-6444 SfuI 7392-7397 SacI 7519-7524 SfuI 7392-7397 SacI 7519-7524 XhoI 7522-7527 StuI 7528-7533 NotI 7535-7540

It should be noted that the proteins encoded by the plant-optimized tcdA (SEQ ID NO:3) and tcbA (SEQ ID NO:5) differ from the native proteins by the addition of an Ala residue at position #2. This modification was made to accommodate the NcoI site which spans the ATG start codon.

The following Table 3 compares the codon composition of the engineered tcdA gene of SEQ ID NO:3 and engineered tcbA gene of SEQ ID NO:5 with the codon compositions of the native genes, the typical dicot genes, and maize genes.

TABLE 3 % in % in SEQ SEQ amino ID % in ID % in % in % in acid codon NO:3 tcdA NO:5 tcbA dicot maize Ala GCT 62 21 69 41 42 24 GCC 26 32 27 17 27 34 GCA 11 25 4 22 25 18 GCG 0 21 0 21 6 24 Arg AGG 48 0 60 2 25 26 CGC 22 36 18 16 11 24 AGA 20 11 15 6 30 15 CGT 11 39 7 57 21 11 CGG 0 7 0 13 4 15 CGA 0 8 0 6 8 9 Asn AAC 100 32 100 33 55 68 AAT 0 68 0 67 45 32 Asp GAC 67 22 70 25 42 63 GAT 33 78 30 75 58 37 Cys TGC 100 30 100 19 56 68 TGT 0 70 0 81 44 32 End TGA 100 0 100 0 33 59 TAG 0 0 0 0 19 21 TAA 0 100 0 100 48 20 Gln CAA 65 61 74 53 59 38 CAG 35 39 26 47 41 62 Glu GAG 100 24 98 36 51 71 GAA 0 76 2 64 49 29 Gly GGT 67 37 64 44 33 20 GGC 32 36 36 22 16 42 GGA 1 20 0 19 38 19 GGG 0 8 0 16 12 20 His CAC 62 40 72 31 46 62 CAT 38 60 28 69 54 38 Ile ATC 73 34 65 24 37 58 ATT 27 51 35 59 45 28 ATA 0 15 0 17 18 14 Leu CTC 54 11 59 7 28 26 TTG 29 17 25 32 26 15 CTT 16 9 15 7 19 17 TTA 0 18 0 19 10 5 CTG 0 32 0 29 9 29 CTA 0 13 0 7 8 8 Lys AAG 99 79 99 75 61 78 AAA 1 21 1 25 39 22 Met ATG 100 100 100 100 100 100 Phe TTC 100 42 100 41 55 71 TTT 0 58 0 59 45 29 Pro CCA 74 30 91 26 42 26 CCT 22 28 7 20 32 22 CCC 4 14 3 7 17 24 CCG 0 27 0 47 9 28 Ser TCC 47 19 55 11 18 23 TCT 35 15 30 15 25 15 AGC 18 22 15 18 18 23 AGT 0 20 0 31 14 9 TCG 0 7 0 8 6 14 TCA 0 17 0 17 19 16 Thr ACC 60 41 64 31 30 37 ACT 28 25 32 34 35 20 ACA 12 21 4 18 27 21 ACG 0 13 0 18 8 22 Trp TGG 100 100 100 100 100 100 Tyr TAC 100 24 100 19 57 73 TAT 0 76 0 81 43 27 Val GTC 69 27 73 11 20 31 GTG 21 17 22 27 29 39 GTT 10 34 3 48 39 21 GTA 0 22 2 14 12 8

EXAMPLE 1

Design Of Plant Codon-Biased Genes Encoding W-14 Peptides TcbA and TcdA

A. Gene Design

The coding strands of the native DNA sequences of the Photorhabdus W-14 genes encoding peptides TcbA and TcdA were scanned for the presence of deleterious sequences such as the Shaw/Kamen RNA destabilizing motif ATTTA, intron splice recognition sites, and poly A addition motifs. This was done using the Macvector Sequence Analysis Software (Oxford Molecular Biology Group, Symantec Corp.), using a custom Nucleic Acid Subsequence File. The native sequence was also searched for runs of 4 or more of the same base.

Motif searching of the native W-14 tcbA and tcdA genes revealed the presence of many potentially deleterious sequences in the protein coding strands, as summarized in Table 4. Not shown, but also present, were many runs of four or more single residues (e.g. the native tcbA gene has 81 runs of four A's).

TABLE 4 Native Poly A Gene ATTTA 5′ Splice 3′ Splice Addition* RNAP II term. tcbA 18 7 17 46 0 tcdA 18 7 13 77 1 *Totals of 16 different motifs.

Analyses of eukaryotic genes and plant genes in particular have shown that CG & TA doublets are underrepresented, while the genes are enriched in CT & TG doublets. The sequences of the hemicot biased genes have accordingly been adjusted to encompass these base compositions and to have G+C compositions of about 53%, similar to-many plant genes. When compared to the native W-14 tcbA and tcdA genes, the plant-biased genes have a much more uniform G+C distribution.

Nucleotide changes to remove potentially deleterious sequences were chosen to simultaneously adjust the codon composition of the coding region to more closely reflect that of plant genes. A framework for these changes was provided by the codon bias tables prepared for maize and dicot genes shown in Table 3.

Comparison of codon compositions of the native W-14 genes to maize and dicot genes revealed that the W-14 genes contain a very different preference set of the degenerate codons for the 18 amino acids for which there is a choice (Table 3). For each of 8 amino acids (Phe, Tyr, Cys, Arg, Asn, Lys, Glu, and Gly) in both W-14 genes, the most abundant codon is different from the preferred codons found in either maize or dicot genes. One might expect that translational difficulties would be encountered in efforts to produce in plants proteins (such as TcbA and TcdA) having high relative amounts of these amino acids from mRNAs having large numbers of nonpreferred codons. There is a marked difference in distribution of the codon compositions specifying the other 10 amino acids. For His, Gln, Ile, Val, and Asp, the dicot-preferred codons are found as the most abundant ones in both W-14 genes. For Leu, Thr, Ser, and Ala, the maize preferred codons are the most abundant codon choices found in the tcdA gene. In contrast, the tcbA gene contains only the CCG (Pro) maize-preferred codon as the highest abundance choice.

In making the codon choices, doublet contents were considered, so that adjacent codons preferably did not form CG or TA doublets (which are underrepresented in eukaryotic genes; 1, 4), while CT or TG doublets (which are enriched in eukaryotic genes ibid.) were created when possible.

Choices were also made to utilize a diversity of codons for Met, Trp, Asn, Asp, Cys, Glu, His, Ile, Lys, Phe, Thr, and Tyr.

The sequences were also designed to encode unique 6-bp recognition sites for restriction enzymes, spaced about every 1200 bp. Finally, an additional codon (GCT; Ala) was inserted at the second position to encode an Nco I recognition site encompassing the ATG (Met) start codon. Additional recognition sites were included after the stop codon to facilitate subsequent cloning steps into expression vectors. These features are set forth above in Tables 1 and 2.

The new tcdA and tcbA genes of SEQ ID NO:3 and SEQ ID NO:4 share 73.5%, and 72.6% identity, respectively, to their native W-14 counterparts (Wisconsin Genetics Computer Group, GAP algorithm).

B. Gene Synthesis

The complete synthesis of the plant codon-biased tcbA and tcdA genes was performed under contract by Operon Technologies, Inc. (OPTI, Alameda, Calif.). Basically, chemically synthesized oligonucleotides of appropriate sequence were assembled into DNA pieces about 500 bases long. These were joined together end-to-end (presumably by means of appropriately placed restriction enzyme sites) into four larger pieces of roughly 2 kilobase pairs (kbp) each; therefore each comprised about ¼ of the entire coding region of the particular gene. DNA sequence of the pieces was confirmed at this step. If mistakes in sequence were present, the appropriate oligonucleotides were re-synthesized, and the assembly process was repeated. Once gene fractional parts were sequence verified, they were assembled in pairs to make the gene halves, and again sequence verified. Finally, the two halves were joined, and the sequences of the junctions between the halves was verified. Therefore, each part of the new gene was sequence verified at least twice.

It should be noted that attempts to express the native tcbA or tcdA genes in standard Escherichia coli cloning strains suggests that production of these proteins is lethal. Lethality problems may be encountered if standard cloning vectors having leaky expression from inherent lacZ promoters are used to assemble these genes.

C. Addition of Endoplasmic Reticulum Targeting Peptide to TcdA Coding Region

It is known to those in the field of plant gene expression that proteins are specifically directed into the endoplasmic reticulum (ER) by means of a short signal peptide which is removed during or after the transport process through the ER membrane. The mature (processed) protein is incorporated into the ER endomembrane or is released into the ER lumen where the transported protein may be uniquely folded (aided by chaperonins), modified by glycosylation, accumulated in the vacuole, or additionally translocated (by secretion). These processes are reviewed by Gomord and Faye [V. Gomord and L. Faye, (1996) Signals and mechanisms involved in intracellular transport of secreted proteins in plants. Plant Physiol. Biochem. 34:165-181] and by Bar-Peled et al. [M. Bar-Peled, D.C. Bassham, and N. V. Raikhel, (1996) Transport of proteins in eukaryotic cells: more questions ahead. Plant Molec. Biology 32:223-249]. It is also known that the subcellular recognition mechanisms for an ER signal peptide are evolutionarily somewhat conserved, since the ER signal for a protein normally produced in monocot (maize) cells is recognized and processed normally by dicot (tobacco) cells. This is exemplified by the maize 15 kDa zein ER signal peptide [L. M. Hoffman, D. D. Donaldson, R. Bookland, K. Rashka, and E. M. Herman, (1987) Synthesis and protein body deposition of maize 15-kd zein in transgenic tobacco seeds. EMBO J. 6:3213-3221, and U.S. Pat. No. 5,589,616]. Further, it is known that the ER signal peptide derived from one protein can direct the translocation of a different protein if it is appropriately attached to the second protein by genetic engineering methods [D.C. Hunt and M. J. Chrispeels, (1991) The signal peptide of a vacuolar protein is necessary and sufficient for the efficient secretion of a cytosolic protein. Plant Physiol. 96:18-25, and Denecke, J., J. Botterman, and R. Deblaere (1990) Protein secretion in plants can occur via a default pathway. Plant Cell 2:51-59]. Therefore, one may expose a protein in vivo to different biochemical environments by directing its accumulation in the cytosol (by not providing a signal peptide sequence), or in the ER/vacuole (by provision of an appropriate signal peptide.)

The ER signal peptide of maize 15 kDa zein proteins is known to comprise the first 20 amino acids encoded by the zein coding region. Two examples of such signal peptides the ER signal peptide of 15 kDa zein from A5707 maize, NCBI Accession # M72708, and the ER signal peptide of 15 kDa zein from Black Mexican Sweet maize, NCBI Accession #M13507. There is only a single amino acid difference (Ser vs Cys at residue 17) between these signal peptides.

SEQ ID NO:5 is a modified sequence coding the ER signal peptide of 15 kDa zein from Black Mexican Sweet maize. The modifications embodied in this sequence were made to accommodate the different monocot/dicot codon usages and other sequence motif considerations discussed above in the design of the plant-optimized tcdA coding region. The sequence includes an additional Ala residue at position #2 to accommodate the NcoI site which spans the ATG start codon.

SEQ ID NO:6 gives a sequence coding for the full-length native TcdA protein (amino acids 22-2537) fused to the modified 15 kDa zein endoplasmic reticulum signal peptide (amino acids 1-21).

EXAMPLE 2 Transformation of Tobacco with Agrobacterium Carrying Plasmid pDAB2041 Encoding Photorhabdus Toxins

A. Plasmid pDAB2041

Preparation of tobacco transformation vectors was accomplished in three steps. First, a modified plant-optimized tcdA coding region was ligated into a tobacco plant expression cassette plasmid. In this step, the coding region was placed under the transcriptional control of a promoter functional in tobacco plant cells. RNA transcription termination and polyadenylation were mediated by a downstream copy of the terminator region from the Agrobacterium nopaline synthase gene. Two plasmids designed to function in this role are pDAB1507 and pDAB2006. In the second step, the complete gene comprised of the promoter, coding region, and terminator region was ligated between the T-DNA borders of an Agrobacterium binary vector, pDAB1542. Also positioned between the T-DNA borders was a plant selectable marker gene to allow selection of transformed tobacco plant cells. In the third step, the engineered binary vector plasmid was conjugated from its E. coli host strain into a disabled Agrobacterium tumefaciens strain capable of transforming tobacco plant cells that regenerate into fertile transgenic plants.

It is a feature of plasmid pDAB1507 that any coding region having an NcoI site at its 5′ end and a SacI site 3′ to the coding region, when cloned into the unique NcoI and SacI sites of pDAB1507, is placed under the transcriptional control of an enhanced version of the CaMV 35S promoter. It is also a feature of pDAB1507 that the 5′ untranslated leader (UTR) sequence preceding the NcoI site comprises a modified version of the 5′ UTR of the MSV coat protein gene, into which has been cloned an internally deleted version of the maize Adh1S intron 1. Additionally it is a feature of pDAB1507 that transcription termination and polyadenylation of the mRNA containing the introduced coding region are mediated by termination/Poly A addition sequences derived from the nopaline synthase (Nos) gene. Finally, it is a feature of pDAB1507 that the entire assembly of promoter/coding region/3′UTR can be obtained as a single DNA fragment by cleavage at the flanking NotI sites.

It is a feature of plasmid pDAB2006 that any coding region having an NcoI site at its 5′ end and a SacI site 3′ to the coding region, when cloned into the unique NcoI and SacI sites of pDAB2006, is placed under the transcriptional control of the CaMV 35S promoter. It is also a feature of pDAB2006 that the 5′ untranslated leader (UTR) sequence preceding the NcoI site comprises a polylinker. Additionally it is a feature of pDAB2006 that transcription termination and polyadenylation of the mRNA containing the introduced coding region are mediated by termination/Poly A addition sequences derived from the nopaline synthase (Nos) gene. Finally, it is a feature of pDAB2006 that the entire assembly of promoter/coding region/3′UTR can be obtained as a single DNA fragment by cleavage at the flanking NotI sites.

It is a feature of pDAB1542 that any DNA fragment flanked by NotI sites can be cloned into the unique NotI site of pDAB1542, thus placing the introduced fragment between the T-DNA borders, and adjacent to the neomycin phosphotransferase II (kanamycin resistance) gene.

To prepare a plant-expressible gene to produce the non-targeted TcdA protein in tobacco plant cells, DNA of a plasmid (pA0H_(—)4-OPTI) containing the plant-optimized tcdA coding region, (SEQ ID No:3) was cleaved with restriction enzymes NcoI and SacI, and the large 7550 bp fragment was ligated to similarly-cut DNA of plasmid pDAB1507 to produce plasmid pDAB2040. DNA of pDAB2040 was then digested with NotI, and the 8884 bp fragment was ligated to NotI digested DNA of pDAB1542 to produce plasmid pDAB2041. This plasmid was then conjugated by triparental mating [Firoozabady, E., D. L. DeBoer, D. J. Merlo, E. L. Halk, L. N. Amerson, K. E. Rashka, and E. E. Murray (1987) Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Molec. Biol. 10:105-116] from the host Escherichia coli strain (XL1-Blue, Stratagene, La Jolla, Calif.), into the nontumorigenic Agrobacterium tumefaciens strain EHA101S, which is a spontaneous streptomycin-resistant mutant of strain EHA101 (Hood, E. E., G. L. Helmer, R. T. Fraley, and M. D. Chilton (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168:1291-1301). Strain EHA101S(pDAB2041) was then used to produce transgenic tobacco plants that expressed the TcdA protein.

B. Plasmid pRK2013

To prepare a plant-expressible gene to produce the endoplasmic reticulum-targeted TcdA protein in tobacco plant cells, DNA of a plasmid (pA0H_(—)4-ER) containing the plant-optimized, ER-targeted tcdA coding region, (SEQ ID No:6) was cleaved with restriction enzymes NcoI and SacI, and the large 7610 bp fragment was ligated to similarly-cut DNA of plasmid pDAB2006 to produce plasmid pDAB1833. DNA of pDAB1833 was then digested with NotI, and the 8822 bp fragment was ligated to NotI digested DNA of pDAB1542 to produce plasmid pDAB2052. This plasmid was then conjugated by triparental mating from the host Escherichia coli strain (XL1-Blue), into the nontumorigenic Agrobacterium tumefaciens strain EHA101S. Strain EHA101S(pDAB2052) was then used to produce transgenic tobacco plants that expressed the TcdA protein containing an amino terminus endoplasmic reticulum targeting peptide.

C. Transfer of Plasmid pDAB2041 Into Agrobacterium Strain EHA101S

Cultures of E. coli carrying the engineered Ti plasmid pDAB2041 (plasmid containing the rebuilt Toxin A gene, tcdA), E. coli carrying the plasmid pRK2013, and Agrobacterium strain EHA101S were grown overnight, then mixed 1:1:1 on plain LB medium solidified with agar and cultured in the dark at 28° C. Two days later, the lawn of bacteria was scraped up with a loop, suspended in plain LB medium, vortexed, and then diluted 1:10⁴, 1:10⁵, and 1:10⁶ fold in plain LB liquid medium. Aliquots of these dilutions were spread on selective plates containing medium YEP plus erythromycin (100 mg/L) and streptomycin (250 mg/L) and grown at 28° C. Two days later, single colonies were picked and streaked onto the same medium, then spread to give single colonies. Single colonies were picked again and streaked, then spread for single colonies. Single colonies were picked a third time, grown as streaks, then subjected to a quality analysis involving growth on lactose medium and chromogenic assay with Benedict's reagent. Of ten strains developed in this way, the fastest coloring colony was chosen for further work.

D. Transformation of Tobacco with Agrobacterium Carrying Plasmid pDAB2041

Tobacco transformation with Agrobacterium tumefaciens was carried out by a method similar, but not identical, to published methods (R Horsch et al, 1988. Plant Molecular Biology Manual, S. Gelvin et al, eds., Kluwer Academic Publishers, Boston). To provide source tissue for the transformation, tobacco seed (Nicotiana tabacum cv. Kentucky 160) were surface sterilized and planted on-the surface of TOB−, which is a hormone-free Murashige and Skoog medium (T. Murashige and F. Skoog, 1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol. 75: 473-497) solidified with agar. Plants were grown for 6-8 weeks in a lighted incubator room at 28-30° C. and leaves were collected sterilely for use in the transformation protocol. Approximately one cm² pieces were sterilely cut from these leaves, excluding the midrib. Cultures of the Agrobacterium strains (EHA101S containing pDAB2041), which had been grown overnight on a rotor at 28° C., were pelleted in a centrifuge and resuspended in sterile Murashige & Skoog salts, adjusted to a final optical density of 0.7 at 600 nm. Leaf pieces were dipped in this bacterial suspension for approximately 30 seconds, then blotted dry on sterile paper towels and placed right side up on medium TOB+ (Murashige and Skoog medium containing 1 mg/L indole acetic acid and 2.5 mg/L benzyladenine) and incubated in the dark at 28° C. Two days later the leaf pieces were moved to medium TOB+ containing 250 mg/L cefotaxime (Agri-Bio, North Miami, Fla.) and 100 mg/L kanamycin sulfate (AgriBio) and incubated at 28-30° C. in the light. Leaf pieces were moved to fresh TOB+ with cefotaxime and kanamycin twice per week for the first two weeks and once per week thereafter. Leaf pieces which showed regrowth of the Agrobacterium strain were moved to medium TOB+ with cefotaxime and kanamycin, plus 100 mg/l carbenicillin (Sigma). Four to six weeks after the leaf pieces were treated with the bacteria, small plants arising from transformed foci were removed from this tissue preparation and planted into medium TOB− containing 250 mg/L cefotaxime and 100 mg/L kanamycin in Magenta GA7 boxes (Magenta Corp., Chicago). These plantlets were grown in a lighted incubator room. After 3-4 weeks the primary transgenic plants had rooted and grown to a size sufficient that leaf samples could be analyzed for expression of protein from the transgene. Twenty-five independent transgenic events were recovered as single plants from the pDAB2041 transformation.

Eight independent lines expressing various levels of transgenic protein from the T-DNA of pDAB2041 were propagated in vitro from leaf pieces as follows. Twelve to sixteen approximately one cm² pieces were sterilely cut from leaves of each primary transgenic plant, excluding the midrib and all naturally occurring edges. These leaf pieces were placed on medium TOB+ containing 250 mg/L cefotaxime and 100 mg/L kanamycin, and cultured in the lighted incubator at 28-30° C. for 3-4 weeks, at which time small plants could be cut from the proliferating tissue mass. Several small plantlets from each transgenic line were moved into Magenta boxes containing medium TOB− plus cefotaxime and kanamycin and allowed to root and grow. The proliferating tissue mass was further cultured on medium TOB+ with cefotaxime and kanamycin, and additional plants could be cut out and grown up as needed.

Plants were moved into the greenhouse by washing the agar from the roots, transplanting into soil in 5½″ square pots, placing the pot into a Ziploc bag (DowBrands), placing plain water into the bottom of the bag, and placing in indirect light in a 30° C. greenhouse for one week. After one week the bag could be opened; the plants were fertilized and allowed to grow further, until the plants were acclimated and the bag was removed. Plants were grown under ordinary warm greenhouse conditions (30° C., 16H light). Plants were suitable for sampling four weeks post transplant.

EXAMPLE 3 Chacterization of Transgenic Tobacco Plants Expressing Photorhabdus Toxin that Confer Insect Control

A. Polyclonal Antibody Production

The E. coli produced recombinant TcdA protein was purified by a series of column purification. The protein was sent to Berkley Antibody Company (Richmond, Calif.) for the production of antiserum in a rabbit. Inoculations with the antigen were initiated with 0.5 mg of protein followed by four boosting injections of 0.25 mg each at about three week intervals. The rabbit serum was tested by the standard Western analysis using the recombinant TcdA protein as the antigen and enhanced chemi-luminescens, ECL method (Amersham, Arlington Heights, Ill.) The antibodies (PAb-EA₀) were purified using a PURE I antibody purification kit (Sigma, St. Luis, Mo.). PAb-EA₀ antibodies recognize the full-length TcdA and its processed components.

B. Expression of TcdA Protein in Tobacco

Protein was extracted from the leaf tissue of transformed and non-transformed tobacco plants following the procedure described immediately below.

Two leaf disks of 1.4 cm in diameter were harvested from the middle portion of a fully expanded leaf. The disks were placed on a 1.6×4 cm piece of 3M Whatman paper. The paper was folded lengthwise and inserted in a flexible straw. Four hundred micro liters of the extraction buffer (9.5 ml of 0.2 M NaH₂PO₄, 15.5 ml of 0.2 M Na₂HPO₄, 2 ml of 0.5 M Na₂EDTA, 100 ml of Triton X100, 1 ml of 10% Sarkosyl, 78 ml of beta-mercaptoethanol, H₂O to bring total volume to 100 ml) was pipetted on to the paper. The straw containing the sample was then passed through a rolling device used for squeezing out the extract 1.5 mL micro centrifuge tube was placed at the other end of the straw to collect the extract. The extract was centrifuged for 10 minutes at 14,000 rpm in an Eppendorf regrigerated microcentrifuge. The supernatant was transferred into a new tube. Protein quantitation analysis was performed using the standard Bio-Rad Protein Analysis protocol (Bio-Rad Laboratories, Hercules, Calif.). The extract was diluted to 2 mg/ml of total protein using the extraction buffer.

For the detection of transgenic protein, Western blot analysis was performed. Following a standard procedure for protein separation (Laemmli, 1970), 40 μg of protein was loaded in each well of 4-20% gradient polyacrylamide gel (Owl Scientific Co., MA) for electrophoresis. Subsequently, the protein was transferred onto a nitrocellulose membrane using a semi-dry electroblotter (Pharmacia LKB Biotechnology, Piscataway, N.J.). The membrane was incubated for one hour in Blotto (5% milk in TBST solution; 25 mM Tris HCL pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20). Thereafter, Blotto was replaced by the primary antibody solution (in Blotto). After one hour in the primary antibody, the membrane was washed with TBST for five minutes three times. Then the secondary antibody in Blotto (1:2000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase; Bio-Rad Laboratories). was added to the membrane. After one hour of incubation, the membrane was washed with an excess amount of TBST for 10 minutes four times. The protein was visualized by using the enhanced chemi-luminescens, ECL method (Amersham, Arlington Heights, Ill.). The differential intensity of the protein bands were measured using densitometer (Molecular Dynamics Inc., Sunnyvale, Calif.).

To determine the expression of TcdA protein in tobacco transformed with pDAB2041, PAb-EA₀ antibodies were used as the primary antibodies. The expression levels of TcdA protein varied among independent transformation events. The primary plant generated from the event #2041-13 showed the highest level of pre-pro TcdA expression of extractable protein. When the leaf pieces from this plant (#2041-13) were used in in vitro propagation, several plants were obtained. Seven of these plants were analyzed for the expression of the TcdA protein. All but one plant produced the full-length TcdA protein as well as some processed peptide components. Using the antibodies specific to Neomycin phospho-transferase, NPT (5 prime-3 prime, Boulder, Colo.), the expression the selectable marker gene (npt II) was detected. Similar results were obtained for #2041-29.

TABLE 5 Western analysis of plants derived from event #2041-13. Plant # TcdA NPT (selectable marker) 2041-13A + not done 2041-13B + not done 2041-13-1 − + 2041-13-2 + + 2041-13-3 + + 2041-13-4 + + 2041-13-5 + +

C. Nucleic Acid Analysis of Transgenic Tobacco Lines

Genomic DNA was prepared from a group of 2041 transgenic events. The lines included Magenta box stage 2041-13, and greenhouse stage plants 2041-13-1, 2041-13-2, 2041-13-5, 2041-9, 2041-20A and 2041-20B. A transgenic GUS line (2023) was included as a negative control. Southern analysis of these lines was performed. The genomic tobacco DNA was restricted with the enzyme SstI which should result in a 8.9 kb hybridization product when hybridized to a tcdA gene specific probe. The 8.9 kb hybridization product should consist of the 35T promoter and the tcdA coding region. All 2041 plants contained a band of the expected size. Events 2041-9 and -20 appear to be the same line with 5 identical hybridizing bands. Event 2041-13 produced 6 hybridization fragments with the tcdA coding region probe. Magenta box and various greenhouse plants of 2041-13 all produced the same hybridization profile. This hybridization pattern was different from that of events 2041-9 and -20.

RNA analysis, using the tcdA coding region probe, was performed on the same group of greenhouse 2041 plants. Immunoblot analysis had revealed that plants 2041-9, 2041-20A, 2041-20B, and 2041-13-1 produced no detectable TcdA protein; while 2041-13-2 and 2041-13-5 produced substantial amounts of full-length TcdA. Northern analysis was in agreement with the immunoblot result. A faint RNA signal was detected for plants 2041-9, 2041-20A, 2041-20B, and 2041-13-1. Only faintly visible was a band corresponding to full-length tcdA transcript in plant 2041-13.1. In contrast, for plants 2041-13-2 and 2041-13-5 a strong RNA signal was detected, with a substantial amount of full-length size (˜8.0 kb) tcdA transcript. These data support the observed bioassay activity for this group of plants.

Genomic DNA was prepared from a second functionally active 2041 transgenic event, 2041-29. Southern analysis of this line was performed. A transgenic GUS line (2023) was included as a negative control, DNA of line 2041-9 was included as a positive control.

The genomic tobacco DNAs were restricted with the enzyme SstI which should result in a 8.9 kb hybridization product when hybridized to a tcdA gene specific probe. The 8.9 kb hybridization product should consist of the 35T promoter and the tcdA coding region. For plant 2041-29-5, three hybridization products larger than 8.9 kb the were detected with the tcdA gene specific probe. Immunoblot analysis has demonstrated pre-pro TcdA protein is made by this plant, it is therefore likely that a restriction site was lost during transformation or regeneration, or the 2041-29 genomic DNA was not thoroughly digested.

D. Tobacco Leaf-Disk Tests with Tobacco Hornworm Exhibiting Insect Control

Leaves were sampled from tobacco plants, Nicotiana tabaco, previously transplanted into the greenhouse. A single leaf was sampled from each plant on each test date. Leaves were selected from the zone where younger elongate leaves transition into older ovate leaves. Excised leaves were placed into 12 oz. cups with the petiole submerged in water to maintain turgor, and transported to the laboratory.

Eight, 1.4 cm disks were cut from the center portion of one side of each leaf (right adaxial side up, with distal portion facing away from the observer). Each disk was placed individually into a well of a C-D International 128 well tray (Pitman, N.J.) into which 0.5 ml of a 1.6% aqueous agar solution had been previously pipetted. The solidified agar prevented the leaf disks from drying out. The adaxial surface of the disk was always oriented up.

A single neonate tobacco hornworm, Manduca sexta, was placed on each disk and the wells were sealed with vented plastic lids. The assay was held at 27° C. and 40% RH. Larval mortality and live-weight data were collected after 3 days. Data were subjected to analysis of variance and Duncan's multiple range test (α=0.05)(Proc GLM, SAS Institute Inc., Cary, N.C.). Data were transformed using a logarithmic function to correct a correlation between the magnitude of the mean and variance.

TABLE 6 Results of leaf-disk assays from greenhouse grown tobacco plants with event 2041-13. Weight of Surviving Larvae (mg) & Duncan's Group¹ Plant 3 Test TRT Plant Age Pretest Test 1 Test 2 Test 3 Sum. 13 non-transformed-2 young — — — 18.8 a* — 14 non-transformed-3 young — — — 17.0 ab — 16 non-transformed-5 young — — — 16.4 ab — 3 2041-13-1(western−) young — 17.6 a 18.2 a 16.1 ab 17.3 a 9 Gus Control old 19.3 a 14.6 a 16.3 a 14.5 ab 15.1 a 10 non-transformed-1 young — 8.3 b 16.8 a 13.9 b 13.0 b 11 2041-20B(western−) old — 10.0 b* 13.7 ab 14.6 ab 12.9 b 15 non-transformed-4 young — — — 13.0 bc — 8 2041-20A(western−) old 15.7 a 8.3 b 11.3 bc 9.2 cd 9.6 c 12 2041-9(western−) old 19.5 a — — 7.9 d — 7 2041-13-5(western+) young — 6.3 bc 9.6 cd 7.2 de 7.7 d 5 2041-13-3(western+) young — 6.4 6.2 e 6.8 de** 6.4 de bc**** 1 2041-13A(western+) old 7.2 b 6.8 bc* 7.0 de* 5.4 e 6.4 de 6 2041-13-4(western+) young — 4.9 c**** 5.8 e 7.6 d 6.4 de 4 2041-13-2(western+) young — 5.7 bc 5.7 e** 7.5 d 6.3 de 2 2041-13B(western +) old — 4.7 c** 5.6 e 7.2 de 5.9 e *Number of stars corresponds to the number of dead larvae per 8 tested. ¹Data transformed (logarithm) for analysis. Means followed by the same letter are not significantly different (alpha = 0.05).

TABLE 7 Results Of Leaf-Disk Assays From Greenhouse Grown Tobacco Plants With Event 2041-29. MEAN WGT (MG)/Duncan's Group Four Test Plant Test 1 Test 2 Test 3 Test 4 Summary 2014-6 GUS 1 15.8 a 16.6a **5.5bc *12.9ab 13.2 a 2014-6 GUS 2 14.4 a *6.6 bc *13.4a 15.2a 12.6 a KY-160 NTC 13.4 a 6.7 bc 7.9b 8.5bc 9.1 b 2041-29 4P *4.9 b *7.3b ****6.9b ******** 6.3 c 2041-29 7 *5.9 b 5.1bc ***6.7b ***7.2c 6.1 c 2041-29 3P *5.6 b **7.9b *****6.5b ***3.6d 5.9 c 2041-29 2P 6.3 b ****4.7c ******4.1c ******4.6d 5.4 c *Number of stars corresponds to the number of dead larvae per 8 tested. ¹Data transformed (logarithm) for analysis. Means followed by the same letter are not significantly different (alpha = 0.05).

All event 2041-29 plants significantly depressed THW larval weight gain compared to control plants. Average weight depression was 49%. Statistically significant mortality occurred in THW larvae exposed to foliage from 2041-29 plants. Mortality averaged 37.5% compared to 5.2% in controls.

E. Isolation and Characterization of Functional Photorhabdus Toxin Protein from Transgenic Plants

Seven grams of transgenic tobacco plants (2041-13) expressing TcdA (Toxin A) gene were homogenized with 10 ml 50 mM Potassium Phosphate buffer, pH 7.0 using a bead beater (Biospec Products, Bartlesville, Okla.) according to manufacturer's instructions. The homogenate was filtered through four layers of cheese cloth and then centrifuged at 35,000 g for 15 min. The supernant was collected and filtered through 0.22 μm Millipore ExpressT™ membrane. It was then applied to a Superdex 200 cloumn (2.6×40 cm) which had been equilibrated with 20 mM Tris buffer, pH 8.0 (Buffer A). The protein was eluted in Buffer A at a flow rate of 3 ml/min. Fractions with 3 ml each were collected and subjected to southern corn rootworm (SCR) bioassay. It was found that fractions corresponding to a native molecular weight around 860 kDa had the highest insecticidal activity. Western analysis of the active fraction using a polyclonal antibody specific to Toxin A indicated the presence of full-length TcdA peptide. The active fractions were further combined and applied to a Mono Q 10/10 column which had been equilibrated with Buffer A. Proteins bound to the column were then eluted by a linear gradient of 0 to 1 M NaCl in Buffer A. Fractions with 2 ml each were collected and analyzed by both SCR bioassay and Western using antibody specific to Toxin A. The results again demonstrated the correlation between insecticidal activity and presence of full-length TcdA peptide.

F. Characterization of Progeny Transgenic Plants

The inheritability of the genetically engineering plants containing the Photorhabdus toxin gene was evaluated by generating Fl progeny. Progeny was generated from 2041-13 event by selfing expression positive plants. The 2041-13 plants in the greenhouse were allowed to self-pollinate. Seed capsules were collected when mature and were allowed to dry and after-ripen on the laboratory bench for two weeks. Seed from plant designated 2041-13A was surface-sterilized and distributed on the surface of medium TOB− without selection, to allow recovery of nonexpressing or nontransgenic progeny as well as expressing and segregating transgenic siblings. Seed was germinated in a C lighted incubator room (16H light, 28 C). After 1 month, fifty-one seedlings, designated 2041-13A-S1 through S51, were distributed into Magenta boxes containing medium TOB− to grow further. Three weeks later, leaf samples from these Magenta-box grown seedlings were submitted for evaluation of the level of expression of TcdA toxin.

Leaf samples were tested for kanamycin response by placing sterile leaf segments on medium TOB+ containing 100 mg/L kanamycin in the light and scoring for tissue growth and color after two weeks. All leaf pieces showed some positive response, indicating complex segregation.

This group of in vitro grown event 2041-13 progeny seedlings were all transplanted into the greenhouse approximately two months after seeding onto medium, using the following method. After washing the agar from the roots,_plants were transplanted into 5½ inch square pots in a soil mix containing 75% MetroMix and 25% mineral soil. They were enclosed in a zip-lock bag and plain water added to leave 1-2 inches of water in the bottom of the bag after soil absorption. These bags were closed and placed under a cart in the greenhouse to protect them from direct sunlight. The bags were opened after 5-6 days, and removed after 7 days, when the plants were adapted to soil and were moved to the top of the cart for normal greenhouse culture. Plants were ready to test in insect bioassays at four weeks post transplant.

F1 progeny were evaluated for expression of protein toxin by immunological screen and for biological activity by plant bioassays, as described previously, using tobacco hornworm. There existed a positive correlation between levels of expression protein toxin and degree of growth inhibition and at higher expression levels mortality was observed. The biological activity was observed to be statistical significance with high cofidence levels between populations of non-transformed and transformed expressing protein toxin.

The following table summarizes the results of insect (tobacco hornworm) bioassays conducted with Fl progeny of self-fertilized 2041-13 plants genetically engineered to produce the “204” A toxin. The tests included 6 non-expressing progeny (protein-negative controls), 45 toxin A expressors, and 4 non-transformed controls (KY-160). Results are from three leaf-disk assays (method previously outlined) where eight disks were used per test. The data were analyzed using analysis of variance and were blocked by test.

The treatment effect for each of these analyses indicated the Pr>F was less than 0.0001. The Toxin A expressors produced significant control of tobacco hornworm compared to each of the control groups based on each of the three measures of efficacy. The two control groups behaved similarly. Statistical analysis using ANOVA and an LSD test with alpha equal to 0.01 (or 1%) showed differences between the 3 groups. The LSD test indicated that the non-expressors and the non-transformed plants were similar in larvae weights but the expressors gave weights significantly lower than either of the other two groups of plants. These data demonstrated that the genetic basis for insect control was inheritable and corresponded to the presence of expressed toxin gene.

TABLE 8 Tobacco hornworm results from F1 progeny of self- fertilized 2041-13 tobacco plants. Mean Value and Duncan's Grouping^(d) Total Weight Survivor Weight Leaf Area Treatment Group (mg)^(a) (mg)^(b) (cm²)^(c) Non-transformed Control 15.8 a 15.8 a 1.2 a Protein-negative Control 16.4 a 16.5 a 1.2 a Toxin A Expressor  8.1 b  9.2 b 4.9 b ^(a)Average insect weight with dead insects considered to weigh nothing. ^(b)Average insect weight with dead insects excluded from analysis. ^(c)Total leaf area remaining per eight leaf disks. Initial area was approximately 12 cm². ^(d)Means followed by the same letter are not significantly different (alpha = 0.05).

EXAMPLE 4 Transformation of Maize with a Vector Carrying Plasmid pDAB1834 Encoding Photorhabdus Toxins

A. Preparation of Maize Transformation Vectors Containing Modified Plant-Optimized tcdA Coding Regions: Plasmid Pdab1834

Preparation of maize transformation vectors was accomplished in two steps. First, a modified plant-optimized tcdA coding region was ligated into a plant expression cassette plasmid. In this step, the coding region was placed under the transcriptional control of a promoter functional in maize plant cells. RNA transcription termination and polyadenylation were mediated by a downstream copy of the terminator region from the Agrobacterium nopaline synthase gene. One plasmid designed to function in this role is pDAB1538. In the second step, the complete gene comprised of the promoter, coding region, and 3′ UTR terminator region was ligated to a plant transformation vector that contained a plant expressible selectable marker gene which allowed the selection of transformed maize plant cells amongst a background of nontransformed cells. An example of such a vector is pDAB367.

It is a feature of plasmid pDAB1538 that any coding region having an NcoI site at its 5′ end and a SacI site 3′ to the coding region, when cloned into the unique NcoI and SacI sites of pDAB1538, is placed under the transcriptional control of the maize ubiquitin1 (ubi1) promoter. It is also a feature of pDAB1538 that the 5′ untranslated leader (UTR) sequence preceding the NcoI site comprises a polylinker. Additionally it is a feature of pDAB1538 that transcription termination and polyadenylation of the mRNA containing the introduced coding region are mediated by termination/Poly A addition sequences derived from the nopaline synthase (Nos) gene. Finally, it is a feature of pDAB1538 that the entire assembly of promoter/coding region/3′UTR can be obtained as a single DNA fragment by cleavage at the flanking NotI sites.

It is a feature of pDAB367 that the phosphinothricin acetyl transferase protein, which has as its substrate phosphinothricin and related compounds, is produced in plant cells through transcription of its coding region mediated by the Cauliflower Mosaic Virus 35S promoter and that termination of transcription plus polyadenylation are mediated by the nopaline synthase terminator region. It is further a feature of pDAB367 that any DNA fragment containing flanking NotI sites can be cloned into the unique NotI site of pDAB367, thus physically linking the introduced DNA fragment to the aforementioned selectable marker gene.

To prepare a maize plant-expressible gene to produce the endoplasmic reticulum-targeted TcdA protein in plant cells, DNA of a plasmid (pA0H_(—)4-ER) containing the plant-optimized, ER-targeted tcdA coding region, (SEQ ID No:6) was cleaved with restriction enzymes NcoI and SacI, and the large 7610 bp fragment was ligated to similarly-cut DNA of plasmid pDAB1538 to produce plasmid pDAB1832. DNA of pDAB1832 was then digested with NotI, and the 9984 bp NotI fragment was ligated into the unique NotI site of pDAB367 to produce plasmid pDAB1834.

It is a feature of plasmids pDAB1834 that the ubi1 and 35S promoters are encoded on the same DNA strand.

B. Transformation and Regeneration of Transgenic Maize Isolates

Type II callus cultures were initiated from immature zygotic embryos of the genotype “Hi-II.” (Armstrong et al, (1991) Maize Genet. Coop. Newslett., 65: 92-93). Embryos were isolated from greenhouse-grown ears from crosses between Hi-II parent A and Hi-II parent B or F₂ embryos derived from a self- or sib-pollination of a Hi-II plant. Immature embryos (1.5 to 3.5 mm) were cultured on initiation medium consisting of N6 salts and vitamins (Chu et al, (1978) The N6 medium and its application to anther culture of cereal crops. Proc. Symp. Plant Tissue Culture, Peking Press, 43-56), 1.0 mg/L 2,4-D, 25 mM L-proline, 100 mg/L casein hydrolysate, 10 mg/L AgNO₃, 2.5 g/L GELRITE (Schweizerhall, South Plainfield, N.J.), and 20 g/L sucrose, with a pH of 5.8. After four to six weeks callus was subcultured onto maintenance medium (initiation medium in which AgNO₃ was omitted and L-proline was reduced to 6 mM). Selection for Type II callus took place for ca. 12-16 weeks.

Plasmid pDAB1834 was transformed into embryogenic callus. For blasting, 140 μg of plasmid DNA was precipitated onto 60 mg of alcohol-rinsed, spherical gold particles (1.5-3.0 μm diameter, Aldrich Chemical Co., Inc., Milwaukee, Wis.) by adding 74 μL of 2.5M CaCl₂H₂O and 30 μL of 0.1M spermidine (free base) to 300 μL of plasmid DNA and H₂O. The solution was immediately vortexed and the DNA-coated gold particles were allowed to settle. The resulting clear supernatant was removed and the gold particles were resuspended in 1 ml of absolute ethanol. This suspension was diluted with absolute ethanol to obtain 15 mg DNA-coated gold/mL.

Approximately 600 mg of embryogenic callus tissue was spread over the surface of Type II callus maintenance medium as described herein lacking casein hydrolysate and L-proline, but supplemented with 0.2 M sorbitol and 0.2 M mannitol as an osmoticum. Following a 4 h pre-treatment, tissue was transferred to culture dishes containing blasting medium (osmotic media solidified with 20 g/L TC agar (PhytoTechnology Laboratories, LLC, Shawnee Mission, Kans.) instead of 7 g/L GELRITE. Helium blasting accelerated suspended DNA-coated gold particles towards and into the prepared tissue targets. The device used was an earlier prototype of that described in U.S. Pat. No. 5,141,131 which is incorporated herein by reference. Tissues were covered with a stainless steel screen (104 μm openings)and placed under a partial vacuum of 25 inches of Hg in the device chamber. The DNA-coated gold particles were further diluted 1:1 with absolute ethanol prior to blasting and were accelerated at the callus targets four times using a helium pressure of 1500 psi, with each blast delivering 20 μL of the DNA/gold suspension. Immediately post-blasting, the tissue was transferred to osmotic media for a 16-24 h recovery period. Afterwards, the tissue was divided into small pieces and transferred to selection medium (maintenance medium lacking casein hydrolysate and L-proline but containing 30 mg/L BASTA® (AgrEvo, Berlin, Germany)). Every four weeks for 3 months, tissue pieces were non-selectively transferred to fresh selection medium. After 7 weeks and up to 22 weeks, callus sectors found proliferating against a background of growth-inhibited tissue were removed and isolated. The resulting BASTA®-resistant tissue was subcultured biweekly onto fresh selection medium. Following western analysis, positive transgenic lines were identified and transferred to regeneration media. Western-negative lines underwent subsequent RNA spot blot analysis to identify negative controls for regeneration.

Regeneration was initiated by transferring callus tissue to cytokinin-based induction medium, which consisted of Murashige and Skoog salts, hereinafter MS salts, and vitamins (Murashige and Skoog, (1962) Physiol. Plant. 15: 473-497) 30 g/L sucrose, 100 mg/L myo-inositol, 30 g/L mannitol, 5 mg/L 6-benzylaminopurine, hereinafter BAP, 0.025 mg/L 2,4-D, 30 mg/L BASTA®, and 2.5 g/L GELRITE at pH 5.7. The cultures were placed in low light (125 ft-candles) for one week followed by one week in high light (325 ft-candles). Following a two week induction period, tissue was non-selectively transferred to hormone-free regeneration medium, which was identical to the induction medium except that it lacked 2,4-D and BAP, and was kept in high light. Small (1.5-3 cm) plantlets were removed and placed in 150×25 mm culture tubes containing SH medium (SH salts and vitamins (Schenk and Hildebrandt, (1972) Can. J. Bot. 50:199-204), 10 g/L sucrose, 100 mg/L myo-inositol, 5 mL/L FeEDTA, and 2.5 g/L GELRITE, pH 5.8). Plantlets were transferred to 12 cm pots containing approximately 0.25 kg of METRO-MIX 360 (The Scotts Co. Marysville, Ohio) in the greenhouse as soon as they exhibited growth and developed a sufficient root system. They were grown with a 16 h photoperiod supplemented by a combination of high pressure sodium and metal halide lamps, and were watered as needed with a combination of three independent Peters Excel fertilizer formulations (Grace-Sierra Horticultural Products Company, Milpitas, Calif.). At the 6-8 leaf stage, plants were transplanted to five gallon pots containing approximately 4 kg METRO-MIX 360, and grown to maturity.

EXAMPLE 5 Characterization Of Transgenic Maize Plants Expressing Photorhabdus Toxin That Confer Insect Control

A. Insect Bioassays

A single leaf was sampled from each plant in each test. Eight, 1.4 cm disks were cut from the outer portion of each leaf (approximately 30 cm long) avoiding the center vein. Each disk was placed individually into a well of a C-D International 128 well tray (Pitman, N.J.) into which 0.5 ml of a 1.6% aqueous agar solution had been previously pipetted. The solidified agar prevented the leaf disks from drying out. The adaxial surface of the disk was always oriented up.

Five neonate southern corn rootworms, Diabrotica undecimpunctata howardi, were placed on each disk and the wells were sealed with vented plastic lids. The assay was held at 27° C. and 40% RH. Larval mortality and live-weight data were collected after 3 days. Data were subjected to analysis of variance and Duncan's multiple range test (α=0.05)(Proc GLM, SAS Institute Inc., Cary, N.C.). Weight data were transformed using a logarithmic function to correct a correlation between the magnitude of the mean and variance.

TABLE 9 Results of Maize Leaf-disk Test vs SCR Mean Survival Mean % Kill Weight (mg) Treatment (Duncan's) (Duncan's) 1834 - 11 68 A 0.064 A 1834 - 17 44 B 0.093 B 1834 - 15 26 BC 0.127 C HiII control 13 C 0.161 C Note: Means followed by the same letter are not significantly different based on Duncan's multiple range test (alpha = 0.05). Insect groups weighing less than 0.1 mg were set to 0.03 mg instead of zero to conduct a more conservative analysis. Mortality (arcsin(sqrt)) and weight(log10) data were transformed for analyses.

The results shown in Table 9 demonstrated that two events expressing TcdA protein were statistically distinct from control lines bioassayed using SCR neonates by mortality and survival weight criteria. These results demonstrated that southern corn rootworm were functionally effected by feeding on maize plants containing and expressing the tcdA gene. Those plants from 1834-11 were used to generate progeny for testing of inheritability of transgene.

B. Production and Progeny Test of tcdA Transgenic Maize

Origin and growth of progeny plants: Sibling plants 1834-11-07 and 1834-11-08, clonally derived by regeneration from the callus of transgenic maize event 1834-11, were transplanted to the greenhouse and pollinated with inbred OQ414. Seeds obtained from these crosses, comprising seed lots 1834-11-07A and 1834-11-08A, were planted in Rootrainers (1½ inch×2 inch×8 inch deep, product #647, C. Hummert Intl., Earth City, Mo.) filled with Metro-Mix 360 soilless mix (Scotts Terra-Lite, available from Hummert Intl.) and top irrigated with Hoagland's nutrient solution. (Hoagland's solution contains 229 ppm nitrogen as nitrate, 24.6 ppm nitrogen as ammonium, 26 ppm P, 157 ppm K, 187 ppm Ca, 49 ppm Mg. and 30 ppm Na.)

Greenhouse conditions for this trial were: 16 hour days, daylight supplemented by metal halide lamps as needed to achieve a minimum of 600 ?Einsteins/cm² PAR, and ambient temperature 30 C days, 22 C nights.

Leaves were sampled for protein determination approximately one week after planting. Leaf bioassays were conducted 2-3 weeks after planting; root bioassays were initiated approximately 3 weeks post planting.

Protein analysis of progeny plants: Protein was extracted from leaf and root samples harvested from transgenic plants, line 1834-11 progenies, and non-transformed plants. Each sample was placed on a 1.6×4 cm piece of 3M Whatman T paper. The paper was folded lengthwise and inserted in a flexible straw. A volume of 350 μl of an extraction buffer (9.5 ml of 0.2 M NaH₂PO₄, 15.5 ml of 0.2 M Na₂HPO₄, 2 ml of 0.5 M Na₂EDTA, 100 ml of Triton X-100, 1 ml of 10% Sarkosyl, 78 ml of beta-mercaptoethanol, H₂O to bring total volume to 100 ml, 50 μg/ml Antipain, 50 μg/ml Leupeptin, 0.1 mM Chymostatin, 5 μg/ml Pepstatin) was pipetted on to the paper. The straw containing the sample was then passed through a rolling device used for squeezing the extract into a 1.5 ml microcentrifuge tube. The extract was centrifuged for 10 minutes at 14,000 rpm in an Eppendorf refrigerated micro-centrifuge. The supernatant was transferred into a new tube. The amount of the total extractable protein was determined using a standard BioRad Protein Analysis protocol (BioRad Laboratories, Hercules, Calif.).

The presence of the TcdA protein was visualized by Western blot analysis following a standard procedure for protein separation (Laemmli, 1970). A volume of twenty μl of extract was loaded in each well of 4-20% gradient polyacrylamide gel (Owl Scientific Co., MA) for electrophoresis. Subsequently, the protein was transferred onto a nitrocellulose membrane using a semi-dry electroblotter (Pharmacia LKB Biotechnology, Piscataway, N.J.). The membrane was incubated for one hour in TBST-M solution (10% milk in TBST solution; 25 mM Tris HCL pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20). Thereafter, the primary antibody (Anti-TcdA in TBST-M) was added. After one hour, the membrane was washed with TBST for five minutes, three times. Then the secondary antibody solution (goat anti-rabbit IgG conjugated to horseradish peroxidase; Bio-Rad Laboratories, in TBST-M) was added to the membrane. After one hour of incubation, the membrane was washed with an excess amount of TBST for 10 minutes, four times. The protein was visualized using the Super Signal® West Pico chemiluminescence method (Pierce Chemical Co., Rockford, Ill.). The protein blot was exposed on a Hyper-film (Amersham, Arlington Heights, Ill.) and was developed within 3 minutes. The intensity of the protein band was measured using a densitometer (Molecular Dynamics Inc., Sunnyvale, Calif.) and compared to standards.

Three of six plants from seed lot 1834-11-07A and three of six plants from seed lot 1834-11-08A produced detectable levels of TcdA protein (Table 1). Approximately 3.8 to 13.3 ppm of TcdA were detected in the leaf blades and 4.1 to 8.4 ppm were detected in the leaf tips of the protein-positive plants. The amounts of TcdA protein detected in the roots were slightly lower than those found in the leaves.

Insect bioassays with progeny plants: Plants were selected for bioassay based on results from Western blot analysis. Twelve (12), 6.4 mm diameter leaf discs were cut from the youngest leaf of each 2 week old seedling. Each disc was placed in a well of a 128-well tray (CD International) containing approximately 0.5 mL of a solidified 2% agar in water solution. Two neonate southern corn rootworm, Diabrotica undecimpunctata howardi (Barber) (SCR), were placed in each well with a leaf disc. Trays were covered with perforated lids and maintained under a controlled environment for 3 days (28 C; 16 hours light:8 hours dark; approx. 60% relative humidity). Living larvae from 4 leaf discs were pooled and weighed producing 3 weight determinations per plant. Average weights were calculated by dividing the pooled weight by the number of survivors. Differences in average weights of SCR fed leaf discs from protein positive and protein negative plants were assessed using analysis of variance on the natural log-transformed average weights (Minitab, v. 12.2, Minitab Inc., State College, Pa.).

Root bioassays were initiated approximately 1 week after the initiation of the leaf disc bioassays. Approximately 24 h prior to eclosion, SCR eggs were suspended in a 0.15% solution of agar in water to a concentration of 100 eggs/ml. Plants were inoculated with SCR eggs by pipetting 2.0 ml of the egg suspension (ie., approximately 200 eggs) just below the soil surface at the base of each plant. Two weeks after inoculation, plants were removed from their Rootrainer pots, their roots washed free of potting mix, and scored for rootworm damage based on a 1 (resistant) to 9 (susceptible) rating system (Welch, 1977). The results of the root ratings were examined using non-parametric tests to determine if the distribution of root ratings from the protein positive plants was the same as the distribution of the ratings from the protein negative plants. Testing was done at the 5% significance level. (StatXact v.3, CYTEL Software Corporation, Cambridge Mass.)

Results from leaf and root bioassays of tcdA protein positive and protein negative progeny plants are summarized in Table 10. The average weights of SCR larvae fed leaf discs from protein positive plants were significantly lower than those of larvae fed leaf discs from protein negative plants (F=4.6; d.f.=1, 34; P≦0.001. The Kolmogorov-Smirnov 2 sample test (p=0.04) and the Wald Wolfowitz runs test (p=0.001) indicated that the protein positive and protein negative root rating distributions were not similar. The Wilcoxon-Mann-Whitney test (p=0.0206) and the Normal Scores test (p=0.206) indicated that the average score for the protein positive plants was lower than the average root rating from the protein negative plants.

TABLE 10 Protein analysis and insect bioassay results with progeny of TcdA transgenic maize. Leaf Disc Root Bioassay Plant TcdA Bioassay Root Rating Number Protein Avg. Wt. (mg) (1-9) 1834-11-07A-30 PRO− 0.190 8 1834-11-08A-21 PRO− 0.196 9 1834-11-08A-16 PRO− 0.195 9 1834-11-08A-14 PRO− 0.137 9 1834-11-07A-22 PRO− 0.208 9 1834-11-07A-20 PRO− 0.175 9 1834-11-07A-26 PRO+ 0.118 9 1834-11-08A-17 PRO+ 0.132 8 1834-11-07A-14 PRO+ 0.110 2 1834-11-07A-11 PRO+ 0.106 4 1834-11-08A-28 PRO+ 0.129 8 1834-11-08A-27 PRO+ 0.108 4

DNA analysis of progeny plants: Leaf samples from 1834-11.7A and 1834-11.8A progeny plants were in conical 50 ml polypropylene tubes and dried in a Labconco Freeze Dry Lyophilizer (Kansas City, Mo.) for 1-2 days. Lyophilized leaves were then ground in a Tecator Cyclotec 1093 Sample mill grinder (Hoganas, Sweden) and stored at −20C. Genomic DNA was extracted by the following procedure: (1) to a 25 ml Conical tube containing 300-500 mg of ground tissue, 9 ml of CTAB (cetyl trimethylammonium bromide solution) was added, and incubated at 65° C. for 1 hour; (2) 4.5 ml of chloroform: octanol (24:1) was added and mixed gently for 5 minutes; (3) samples were centrifuged at 2000 rpm and DNA was precipitated from the supernatant with an equal volume of isopropanol; (4) DNA was collected on a glass hook, washed in ethanol, and dissolved in TE (10 mM Tris.HCl, 0.5 mM EDTA, pH 8.0).

Genomic DNA was digested at 37° C. for 2 hours in an Eppendorf tube containing the following mixture: 8 μl of 800 ug/ml DNA, 2 μl 1 mg/ml BSA (Bovine serum albumin),2 μl 10× buffer, 1 μl SacI, 1 μl EcoRI, and 6 μl H₂O. Digested DNA samples were electrophoresed overnight at 40 mA in a 0.85% SeaKem LE agarose gel(FMC, Rockland, Me.). The gel was blotted onto Millipore Immobilon-Ny+ (Bedford, Mass.) membrane overnight in 20×SSC (NaCl 175.2 g/l, Na citrate 88 g/l). The probe DNA was cut with BamHI/SacI (NEB, Beverly, Mass.) from pDAB1551 plasmid, which released a 7356 bp fragment containing the open reading frame of the rebuilt tcdA gene. This 7356 bp fragment was labeled with P32 using a Stratagene Prime-it RmT dCTP-Labeling Reactions kit (La Jolla, Calif.) and used for Southern hybridization. Hybridization was conducted in hybridization buffer (10% polyethylene glycol, 7% SDS [Sodium dodecyl sulfate], 0.6×SSC, 10 mM NaPO₄, 5 mM EDTA, 10 μg/ml denatured salmon sperm) at 60° C. overnight. After hybridization, the membrane was washed with 10×SSC plus 0.1% SDS at 60° C. for 30 min and exposed to X ray film (Hyperfilm® MP, Amershan Life Sciences, Piscataway, N.J.) for 1-2 days.

Results summarized indicate that a pattern of 8 hybridizing bands (the size of the expected fragment and larger) cosegregated with protein expression in 50% of all progeny assayed. These results are characteristic of a complex insertion at a single site. All seedlings containing the insert also expressed toxin protein.

EXAMPLE 6 Transformation of Rice with a Vector Carrying Plasmid pDAB1553 Encoding Photorhabdus Toxins

A. Plasmid pDAB1553

Plasmid pDAB1553 containing tcdA driven by the maize ubiquitin1 promoter and hpt (hygromycin phosphotransferase providing resistance to the antibiotic hygromycin) under the control of 35T (a modified 35S promoter), was used for transformation.

Preparation of rice transformation vectors was accomplished in two steps. First, a modified plant-optimized tcdA coding region was ligated into a rice plant expression cassette plasmid. In this step, the coding region was placed under the transcriptional control of a promoter functional in plant cells. RNA transcription termination and polyadenylation were mediated by a downstream copy of the terminator region from the Agrobacterium nopaline synthase gene. One plasmid designed to function in this role is plasmid pDAB1538 (described in the section on maize transformation vectors). In the second step, the complete gene comprised of the promoter, coding region, and terminator region was ligated to a rice plant transformation vector that contained a plant expressible selectable marker gene which allowed the selection of transformed rice plant cells amongst a background of nontransformed cells. An example of such a vector is pDAB354-Not1.

It is a feature of pDAB354-Not1 that the hygromycin phosphotransferase protein, which has as its substrate hygromycin B and related compounds, is produced in plant cells through transcription of its coding region mediated by the Cauliflower Mosaic Virus 35S promoter and that termination of transcription plus polyadenylation are mediated by the nopaline synthase terminator region. It is further a feature of pDAB354-Not1 that any DNA fragment containing flanking NotI sites can be cloned into the unique NotI site of pDAB354-Not1, thus physically linking the introduced DNA fragment to the aforementioned selectable marker gene.

To prepare a plant-expressible gene to produce the non-targeted TcdA protein in rice plant cells, DNA of a plasmid (pAOH_(—)4-OPTI) containing the plant-optimized tcdA coding region, (SEQ ID No:3) was cleaved with restriction enzymes NcoI and SacI, and the large 7550 bp fragment was ligated to similarly-cut DNA of plasmid pDAB1538 to produce plasmid pDAB1551. DNA of pDAB1551 was then digested with NotI, and the large 9933 bp fragment was ligated to NotI digested DNA of pDAB354-NotI to produce plasmid pDAB1553.

It is a feature of plasmid pDAB1553 that the ubil and 35S promoters are encoded on the same DNA strand.

B. Production of Rice Transgenics

For initiation of embryogenic callus, mature seeds of a Japonica cultivar, Taipei 309 were dehusked and surface-sterilized in 70% ethanol for 2-5 min. followed by a 30-45 min soak in 50% commercial bleach (2.6% sodium hypochlorite) with a few drops of ‘Liquinox’ soap. The seeds were then rinsed 3 times in sterile distilled water and placed on filter paper before transferring to ‘callus induction’ medium (i.e., NB). The NB medium consisted of N6 macro elements (Chu, 1978, The N6 medium and its application to anther culture of cereal crops. Proc. Symp. Plant Tissue Culture, Peking Press, p43-56), B5 micro elements and vitamins (Gamborg et al., 1968, Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-158), 300 mg/L casein hydrolysate, 500 mg/L L-proline, 500 mg/L L-glutamine, 30 g/L sucrose, 2 mg/L 2,4-dichloro-phenoxyacetic acid (2,4-D), and 2.5 g/L gelrite (Schweizerhall, N.J.) with the pH adjusted to 5.8. The mature seed cultured on ‘induction’ media were incubated in the dark at 28° C. After 3 weeks of culture, the emerging primary callus induced from the scutellar region of mature embryo was transferred to fresh NB medium for further maintenance.

About 140 μg of plasmid pDAB1553 DNA was precipitated onto 60 mg of 1.0 micron (Bio-Rad) gold particles as described herein.

For helium blasting, actively growing embryogenic callus cultures, 2-4 mm in size, were subjected to a high osmoticum treatment. This treatment included placing of callus on NB medium with 0.2 M mannitol and 0.2 M sorbitol (Vain et al., 1993, Osmoticum treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep. 12: 84-88) for 4 h before helium blasting. Following osmoticum treatment, callus cultures were transferred to ‘blasting’ medium (NB+2% agar) and covered with a stainless steel screen (230 micron). The callus cultures were blasted at 2,000 psi helium pressures twice per target. After blasting, callus was transferred back to the media with high osmoticum overnight before placing on selection medium, which consisted NB medium with 30 mg/L hygromycin. After 2 weeks, the cultures were transferred to fresh selection medium with a higher concentration of selection agent, i.e., NB+50 mg/L hygromycin (Li et al., 1993, An improved rice transformation system using the biolistic method. Plant Cell Rep. 12: 250-255).

Compact, white-yellow, embryogenic callus cultures, recovered on NB+50 mg/L hygromycin, were regenerated by transferring to ‘pre-regeneration’ (PR) medium+50 mg/L hygromycin. The PR medium consisted of NB medium with 2 mg/L benzyl aminopurine (BAP), 1 mg/L naphthalene acetic acid (NAA), and 5 mg/L abscisic acid (ABA). After 2 weeks of culture in the dark, they were transferred to ‘regeneration’ (RN) medium. The composition of RN medium is NB medium with 3 mg/L BAP, and 0.5 mg/L NAA. The cultures on RN medium were incubated for 2 weeks at 28° C. under high fluorescent light (325-ft-candles). The plantlets with 2 cm shoot were transferred to ½ MS medium (Murashige and Skoog, 1962, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant.15:473-497) with ½ B5 vitamins, 10 g/L sucrose, 0.05 mg/L NAA, 50 mg/L hygromycin and 2.5 g/L gelrite adjusted to pH 5.8 in magenta boxes. When plantlets were established with well-developed root systems, they were transferred to soil (1 metromix: 1 top soil) and raised in the greenhouse (29/24° C. day/night cycle, 50-60% humidity, 12 h photoperiod) until maturity.

EXAMPLE 7

Chacterization of Transgenic Rice Plants Expressing Photorhabdus Toxin that Confer Insect Control.

A. Insect Bioassays

Insect bioassays were performed using leaf discs and shown to be highly effective in controlling Southern corn rootworm. Diabrotica undecimpunctata howardi eggs are obtained from French Ag Research and hatched in petri dishes held at 28.5° C. and 40% RH. The aerial parts are sampled from the transgenic plants and placed, singly into inverted petri dishes (100×15 mm) containing 15 ml of 1.6% aqueous agar in the bottom to provide humidity and filter paper in the top to absorb condensation. These preparations are infested with five neonate larvae per dish and held at 28.5° C. and 40% RH for 3 days. Mortality and larval weights are recorded. Weight data were transformed using a logarithmic function to correct a correlation between the magnitude of the mean and variance.

TABLE 11 Average Survivor Presence TcdA Weight in mg¹ greenhouse-grown (Duncan's plants (number of +/ Treatment Grouping) number of plants tested) GUS 0.390 A − Control 1553-33 0.170 BCD ++ 1553-44 0.167 BCD +++ 1553-62 0.125 CD +++ 1553-41 0.100 D +++ Note: Means followed by the same letter are not significantly different based on Duncan's multiple range test (alpha = 0.05).

Insect groups weighing less than 0.1 mg were set to 0.03 mg instead of zero to conduct a more conservative analysis. Weight data were transformed (Log10) for analyses. A single replicate was used on each of three test dates. Plants were sampled from magenta boxes.

The results demonstrate that in leaf disc bioassays, several rice events derived by transformation with tcdA gene were demonstrated to statistically have a functional affect on corn rootworm neonate.

                   #             SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 6 <210> SEQ ID NO 1 <211> LENGTH: 7551 <212> TYPE: DNA <213> ORGANISM: Photorhabdus luminescens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(7548) <400> SEQUENCE: 1 atg aac gag tct gta aaa gag ata cct gat gt #a tta aaa agc cag tgt       48 Met Asn Glu Ser Val Lys Glu Ile Pro Asp Va #l Leu Lys Ser Gln Cys   1               5  #                 10  #                 15 ggt ttt aat tgt ctg aca gat att agc cac ag #c tct ttt aat gaa ttt       96 Gly Phe Asn Cys Leu Thr Asp Ile Ser His Se #r Ser Phe Asn Glu Phe              20      #             25      #             30 cgc cag caa gta tct gag cac ctc tcc tgg tc #c gaa aca cac gac tta      144 Arg Gln Gln Val Ser Glu His Leu Ser Trp Se #r Glu Thr His Asp Leu          35          #         40          #         45 tat cat gat gca caa cag gca caa aag gat aa #t cgc ctg tat gaa gcg      192 Tyr His Asp Ala Gln Gln Ala Gln Lys Asp As #n Arg Leu Tyr Glu Ala      50              #     55              #     60 cgt att ctc aaa cgc gcc aat ccc caa tta ca #a aat gcg gtg cat ctt      240 Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gl #n Asn Ala Val His Leu  65                  # 70                  # 75                  # 80 gcc att ctc gct ccc aat gct gaa ctg ata gg #c tat aac aat caa ttt      288 Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gl #y Tyr Asn Asn Gln Phe                  85  #                 90  #                 95 agc ggt aga gcc agt caa tat gtt gcg ccg gg #t acc gtt tct tcc atg      336 Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gl #y Thr Val Ser Ser Met             100       #           105       #           110 ttc tcc ccc gcc gct tat ttg act gaa ctt ta #t cgt gaa gca cgc aat      384 Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Ty #r Arg Glu Ala Arg Asn         115           #       120           #       125 tta cac gca agt gac tcc gtt tat tat ctg ga #t acc cgc cgc cca gat      432 Leu His Ala Ser Asp Ser Val Tyr Tyr Leu As #p Thr Arg Arg Pro Asp     130               #   135               #   140 ctc aaa tca atg gcg ctc agt cag caa aat at #g gat ata gaa tta tcc      480 Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Me #t Asp Ile Glu Leu Ser 145                 1 #50                 1 #55                 1 #60 aca ctc tct ttg tcc aat gag ctg tta ttg ga #a agc att aaa act gaa      528 Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Gl #u Ser Ile Lys Thr Glu                 165   #               170   #               175 tct aaa ctg gaa aac tat act aaa gtg atg ga #a atg ctc tcc act ttc      576 Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Gl #u Met Leu Ser Thr Phe             180       #           185       #           190 cgt cct tcc ggc gca acg cct tat cat gat gc #t tat gaa aat gtg cgt      624 Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Al #a Tyr Glu Asn Val Arg         195           #       200           #       205 gaa gtt atc cag cta caa gat cct gga ctt ga #g caa ctc aat gca tca      672 Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Gl #u Gln Leu Asn Ala Ser     210               #   215               #   220 ccg gca att gcc ggg ttg atg cat caa gcc tc #c cta ttg ggt att aac      720 Pro Ala Ile Ala Gly Leu Met His Gln Ala Se #r Leu Leu Gly Ile Asn 225                 2 #30                 2 #35                 2 #40 gct tca atc tcg cct gag cta ttt aat att ct #g acg gag gag att acc      768 Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Le #u Thr Glu Glu Ile Thr                 245   #               250   #               255 gaa ggt aat gct gag gaa ctt tat aag aaa aa #t ttt ggt aat atc gaa      816 Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys As #n Phe Gly Asn Ile Glu             260       #           265       #           270 ccg gcc tca ttg gct atg ccg gaa tac ctt aa #a cgt tat tat aat tta      864 Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Ly #s Arg Tyr Tyr Asn Leu         275           #       280           #       285 agc gat gaa gaa ctt agt cag ttt att ggt aa #a gcc agc aat ttt ggt      912 Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Ly #s Ala Ser Asn Phe Gly     290               #   295               #   300 caa cag gaa tat agt aat aac caa ctt att ac #t ccg gta gtc aac agc      960 Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Th #r Pro Val Val Asn Ser 305                 3 #10                 3 #15                 3 #20 agt gat ggc acg gtt aag gta tat cgg atc ac #c cgc gaa tat aca acc     1008 Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Th #r Arg Glu Tyr Thr Thr                 325   #               330   #               335 aat gct tat caa atg gat gtg gag cta ttt cc #c ttc ggt ggt gag aat     1056 Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pr #o Phe Gly Gly Glu Asn             340       #           345       #           350 tat cgg tta gat tat aaa ttc aaa aat ttt ta #t aat gcc tct tat tta     1104 Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Ty #r Asn Ala Ser Tyr Leu         355           #       360           #       365 tcc atc aag tta aat gat aaa aga gaa ctt gt #t cga act gaa ggc gct     1152 Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Va #l Arg Thr Glu Gly Ala     370               #   375               #   380 cct caa gtc aat ata gaa tac tcc gca aat at #c aca tta aat acc gct     1200 Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Il #e Thr Leu Asn Thr Ala 385                 3 #90                 3 #95                 4 #00 gat atc agt caa cct ttt gaa att ggc ctg ac #a cga gta ctt cct tcc     1248 Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Th #r Arg Val Leu Pro Ser                 405   #               410   #               415 ggt tct tgg gca tat gcc gcc gca aaa ttt ac #c gtt gaa gag tat aac     1296 Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Th #r Val Glu Glu Tyr Asn             420       #           425       #           430 caa tac tct ttt ctg cta aaa ctt aac aag gc #t att cgt cta tca cgt     1344 Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Al #a Ile Arg Leu Ser Arg         435           #       440           #       445 gcg aca gaa ttg tca ccc acg att ctg gaa gg #c att gtg cgc agt gtt     1392 Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gl #y Ile Val Arg Ser Val     450               #   455               #   460 aat cta caa ctg gat atc aac aca gac gta tt #a ggt aaa gtt ttt ctg     1440 Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Le #u Gly Lys Val Phe Leu 465                 4 #70                 4 #75                 4 #80 act aaa tat tat atg cag cgt tat gct att ca #t gct gaa act gcc ctg     1488 Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile Hi #s Ala Glu Thr Ala Leu                 485   #               490   #               495 ata cta tgc aac gcg cct att tca caa cgt tc #a tat gat aat caa cct     1536 Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Se #r Tyr Asp Asn Gln Pro             500       #           505       #           510 agc caa ttt gat cgc ctg ttt aat acg cca tt #a ctg aac gga caa tat     1584 Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Le #u Leu Asn Gly Gln Tyr         515           #       520           #       525 ttt tct acc ggc gat gag gag att gat tta aa #t tca ggt agc acc ggc     1632 Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu As #n Ser Gly Ser Thr Gly     530               #   535               #   540 gat tgg cga aaa acc ata ctt aag cgt gca tt #t aat att gat gat gtc     1680 Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Ph #e Asn Ile Asp Asp Val 545                 5 #50                 5 #55                 5 #60 tcg ctc ttc cgc ctg ctt aaa att acc gac ca #t gat aat aaa gat gga     1728 Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp Hi #s Asp Asn Lys Asp Gly                 565   #               570   #               575 aaa att aaa aat aac cta aag aat ctt tcc aa #t tta tat att gga aaa     1776 Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser As #n Leu Tyr Ile Gly Lys             580       #           585       #           590 tta ctg gca gat att cat caa tta acc att ga #t gaa ctg gat tta tta     1824 Leu Leu Ala Asp Ile His Gln Leu Thr Ile As #p Glu Leu Asp Leu Leu         595           #       600           #       605 ctg att gcc gta ggt gaa gga aaa act aat tt #a tcc gct atc agt gat     1872 Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Le #u Ser Ala Ile Ser Asp     610               #   615               #   620 aag caa ttg gct acc ctg atc aga aaa ctc aa #t act att acc agc tgg     1920 Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu As #n Thr Ile Thr Ser Trp 625                 6 #30                 6 #35                 6 #40 cta cat aca cag aag tgg agt gta ttc cag ct #a ttt atc atg acc tcc     1968 Leu His Thr Gln Lys Trp Ser Val Phe Gln Le #u Phe Ile Met Thr Ser                 645   #               650   #               655 acc agc tat aac aaa acg cta acg cct gaa at #t aag aat ttg ctg gat     2016 Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Il #e Lys Asn Leu Leu Asp             660       #           665       #           670 acc gtc tac cac ggt tta caa ggt ttt gat aa #a gac aaa gca gat ttg     2064 Thr Val Tyr His Gly Leu Gln Gly Phe Asp Ly #s Asp Lys Ala Asp Leu         675           #       680           #       685 cta cat gtc atg gcg ccc tat att gcg gcc ac #c ttg caa tta tca tcg     2112 Leu His Val Met Ala Pro Tyr Ile Ala Ala Th #r Leu Gln Leu Ser Ser     690               #   695               #   700 gaa aat gtc gcc cac tcg gta ctc ctt tgg gc #a gat aag tta cag ccc     2160 Glu Asn Val Ala His Ser Val Leu Leu Trp Al #a Asp Lys Leu Gln Pro 705                 7 #10                 7 #15                 7 #20 ggc gac ggc gca atg aca gca gaa aaa ttc tg #g gac tgg ttg aat act     2208 Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Tr #p Asp Trp Leu Asn Thr                 725   #               730   #               735 aag tat acg ccg ggt tca tcg gaa gcc gta ga #a acg cag gaa cat atc     2256 Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Gl #u Thr Gln Glu His Ile             740       #           745       #           750 gtt cag tat tgt cag gct ctg gca caa ttg ga #a atg gtt tac cat tcc     2304 Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Gl #u Met Val Tyr His Ser         755           #       760           #       765 acc ggc atc aac gaa aac gcc ttc cgt cta tt #t gtg aca aaa cca gag     2352 Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Ph #e Val Thr Lys Pro Glu     770               #   775               #   780 atg ttt ggc gct gca act gga gca gcg ccc gc #g cat gat gcc ctt tca     2400 Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Al #a His Asp Ala Leu Ser 785                 7 #90                 7 #95                 8 #00 ctg att atg ctg aca cgt ttt gcg gat tgg gt #g aac gca cta ggc gaa     2448 Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Va #l Asn Ala Leu Gly Glu                 805   #               810   #               815 aaa gcg tcc tcg gtg cta gcg gca ttt gaa gc #t aac tcg tta acg gca     2496 Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Al #a Asn Ser Leu Thr Ala             820       #           825       #           830 gaa caa ctg gct gat gcc atg aat ctt gat gc #t aat ttg ctg ttg caa     2544 Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Al #a Asn Leu Leu Leu Gln         835           #       840           #       845 gcc agt att caa gca caa aat cat caa cat ct #t ccc cca gta act cca     2592 Ala Ser Ile Gln Ala Gln Asn His Gln His Le #u Pro Pro Val Thr Pro     850               #   855               #   860 gaa aat gcg ttc tcc tgt tgg aca tct atc aa #t act atc ctg caa tgg     2640 Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile As #n Thr Ile Leu Gln Trp 865                 8 #70                 8 #75                 8 #80 gtt aat gtc gca caa caa ttg aat gtc gcc cc #a cag ggc gtt tcc gct     2688 Val Asn Val Ala Gln Gln Leu Asn Val Ala Pr #o Gln Gly Val Ser Ala                 885   #               890   #               895 ttg gtc ggg ctg gat tat att caa tca atg aa #a gag aca ccg acc tat     2736 Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Ly #s Glu Thr Pro Thr Tyr             900       #           905       #           910 gcc cag tgg gaa aac gcg gca ggc gta tta ac #c gcc ggg ttg aat tca     2784 Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Th #r Ala Gly Leu Asn Ser         915           #       920           #       925 caa cag gct aat aca tta cac gct ttt ctg ga #t gaa tct cgc agt gcc     2832 Gln Gln Ala Asn Thr Leu His Ala Phe Leu As #p Glu Ser Arg Ser Ala     930               #   935               #   940 gca tta agc acc tac tat atc cgt caa gtc gc #c aag gca gcg gcg gct     2880 Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Al #a Lys Ala Ala Ala Ala 945                 9 #50                 9 #55                 9 #60 att aaa agc cgt gat gac ttg tat caa tac tt #a ctg att gat aat cag     2928 Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Le #u Leu Ile Asp Asn Gln                 965   #               970   #               975 gtt tct gcg gca ata aaa acc acc cgg atc gc #c gaa gcc att gcc agt     2976 Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Al #a Glu Ala Ile Ala Ser             980       #           985       #           990 att caa ctg tac gtc aac cgg gca ttg gaa aa #t gtg gaa gaa aat gcc     3024 Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu As #n Val Glu Glu Asn Ala         995           #       1000           #      1005 aat tcg ggg gtt atc agc cgc caa ttc ttt at #c gac tgg gac aaa tac     3072 Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Il #e Asp Trp Asp Lys Tyr     1010              #   1015               #  1020 aat aaa cgc tac agc act tgg gcg ggt gtt tc #t caa tta gtt tac tac     3120 Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Se #r Gln Leu Val Tyr Tyr 1025                1030 #                1035  #               1040 ccg gaa aac tat att gat ccg acc atg cgt at #c gga caa acc aaa atg     3168 Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Il #e Gly Gln Thr Lys Met                 1045  #               1050   #              1055 atg gac gca tta ctg caa tcc gtc agc caa ag #c caa tta aac gcc gat     3216 Met Asp Ala Leu Leu Gln Ser Val Ser Gln Se #r Gln Leu Asn Ala Asp             1060      #           1065       #          1070 acc gtc gaa gat gcc ttt atg tct tat ctg ac #a tcg ttt gaa caa gtg     3264 Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Th #r Ser Phe Glu Gln Val         1075          #       1080           #      1085 gct aat ctt aaa gtt att agc gca tat cac ga #t aat att aat aac gat     3312 Ala Asn Leu Lys Val Ile Ser Ala Tyr His As #p Asn Ile Asn Asn Asp     1090              #   1095               #  1100 caa ggg ctg acc tat ttt atc gga ctc agt ga #a act gat gcc ggt gaa     3360 Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Gl #u Thr Asp Ala Gly Glu 1105                1110 #                1115  #               1120 tat tat tgg cgc agt gtc gat cac agt aaa tt #c aac gac ggt aaa ttc     3408 Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Ph #e Asn Asp Gly Lys Phe                 1125  #               1130   #              1135 gcg gct aat gcc tgg agt gaa tgg cat aaa at #t gat tgt cca att aac     3456 Ala Ala Asn Ala Trp Ser Glu Trp His Lys Il #e Asp Cys Pro Ile Asn             1140      #           1145       #          1150 cct tat aaa agc act atc cgt cca gtg ata ta #t aaa tcc cgc ctg tat     3504 Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Ty #r Lys Ser Arg Leu Tyr         1155          #       1160           #      1165 ctg ctc tgg ttg gaa caa aag gag atc acc aa #a cag aca gga aat agt     3552 Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Ly #s Gln Thr Gly Asn Ser     1170              #   1175               #  1180 aaa gat ggc tat caa act gaa acg gat tat cg #t tat gaa cta aaa ttg     3600 Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Ar #g Tyr Glu Leu Lys Leu 1185                1190 #                1195  #               1200 gcg cat atc cgc tat gat ggc act tgg aat ac #g cca atc acc ttt gat     3648 Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Th #r Pro Ile Thr Phe Asp                 1205  #               1210   #              1215 gtc aat aaa aaa ata tcc gag cta aaa ctg ga #a aaa aat aga gcg ccc     3696 Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Gl #u Lys Asn Arg Ala Pro             1220      #           1225       #          1230 gga ctc tat tgt gcc ggt tat caa ggt gaa ga #t acg ttg ctg gtg atg     3744 Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu As #p Thr Leu Leu Val Met         1235          #       1240           #      1245 ttt tat aac caa caa gac aca cta gat agt ta #t aaa aac gct tca atg     3792 Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Ty #r Lys Asn Ala Ser Met     1250              #   1255               #  1260 caa gga cta tat atc ttt gct gat atg gca tc #c aaa gat atg acc cca     3840 Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Se #r Lys Asp Met Thr Pro 1265                1270 #                1275  #               1280 gaa cag agc aat gtt tat cgg gat aat agc ta #t caa caa ttt gat acc     3888 Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Ty #r Gln Gln Phe Asp Thr                 1285  #               1290   #              1295 aat aat gtc aga aga gtg aat aac cgc tat gc #a gag gat tat gag att     3936 Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Al #a Glu Asp Tyr Glu Ile             1300      #           1305       #          1310 cct tcc tcg gta agt agc cgt aaa gac tat gg #t tgg gga gat tat tac     3984 Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gl #y Trp Gly Asp Tyr Tyr         1315          #       1320           #      1325 ctc agc atg gta tat aac gga gat att cca ac #t atc aat tac aaa gcc     4032 Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Th #r Ile Asn Tyr Lys Ala     1330              #   1335               #  1340 gca tca agt gat tta aaa atc tat atc tca cc #a aaa tta aga att att     4080 Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pr #o Lys Leu Arg Ile Ile 1345                1350 #                1355  #               1360 cat aat gga tat gaa gga cag aag cgc aat ca #a tgc aat ctg atg aat     4128 His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gl #n Cys Asn Leu Met Asn                 1365  #               1370   #              1375 aaa tat ggc aaa cta ggt gat aaa ttt att gt #t tat act agc ttg ggg     4176 Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Va #l Tyr Thr Ser Leu Gly             1380      #           1385       #          1390 gtc aat cca aat aac tcg tca aat aag ctc at #g ttt tac ccc gtc tat     4224 Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Me #t Phe Tyr Pro Val Tyr         1395          #       1400           #      1405 caa tat agc gga aac acc agt gga ctc aat ca #a ggg aga cta cta ttc     4272 Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gl #n Gly Arg Leu Leu Phe     1410              #   1415               #  1420 cac cgt gac acc act tat cca tct aaa gta ga #a gct tgg att cct gga     4320 His Arg Asp Thr Thr Tyr Pro Ser Lys Val Gl #u Ala Trp Ile Pro Gly 1425                1430 #                1435  #               1440 gca aaa cgt tct cta acc aac caa aat gcc gc #c att ggt gat gat tat     4368 Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Al #a Ile Gly Asp Asp Tyr                 1445  #               1450   #              1455 gct aca gac tct ctg aat aaa ccg gat gat ct #t aag caa tat atc ttt     4416 Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Le #u Lys Gln Tyr Ile Phe             1460      #           1465       #          1470 atg act gac agt aaa ggg act gct act gat gt #c tca ggc cca gta gag     4464 Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Va #l Ser Gly Pro Val Glu         1475          #       1480           #      1485 att aat act gca att tct cca gca aaa gtt ca #g ata ata gtc aaa gcg     4512 Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gl #n Ile Ile Val Lys Ala     1490              #   1495               #  1500 ggt ggc aag gag caa act ttt acc gca gat aa #a gat gtc tcc att cag     4560 Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Ly #s Asp Val Ser Ile Gln 1505                1510 #                1515  #               1520 cca tca cct agc ttt gat gaa atg aat tat ca #a ttt aat gcc ctt gaa     4608 Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gl #n Phe Asn Ala Leu Glu                 1525  #               1530   #              1535 ata gac ggt tct ggt ctg aat ttt att aac aa #c tca gcc agt att gat     4656 Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn As #n Ser Ala Ser Ile Asp             1540      #           1545       #          1550 gtt act ttt acc gca ttt gcg gag gat ggc cg #c aaa ctg ggt tat gaa     4704 Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Ar #g Lys Leu Gly Tyr Glu         1555          #       1560           #      1565 agt ttc agt att cct gtt acc ctc aag gta ag #t acc gat aat gcc ctg     4752 Ser Phe Ser Ile Pro Val Thr Leu Lys Val Se #r Thr Asp Asn Ala Leu     1570              #   1575               #  1580 acc ctg cac cat aat gaa aat ggt gcg caa ta #t atg caa tgg caa tcc     4800 Thr Leu His His Asn Glu Asn Gly Ala Gln Ty #r Met Gln Trp Gln Ser 1585                1590 #                1595  #               1600 tat cgt acc cgc ctg aat act cta ttt gcc cg #c cag ttg gtt gca cgc     4848 Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Ar #g Gln Leu Val Ala Arg                 1605  #               1610   #              1615 gcc acc acc gga atc gat aca att ctg agt at #g gaa act cag aat att     4896 Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Me #t Glu Thr Gln Asn Ile             1620      #           1625       #          1630 cag gaa ccg cag tta ggc aaa ggt ttc tat gc #t acg ttc gtg ata cct     4944 Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Al #a Thr Phe Val Ile Pro         1635          #       1640           #      1645 ccc tat aac cta tca act cat ggt gat gaa cg #t tgg ttt aag ctt tat     4992 Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Ar #g Trp Phe Lys Leu Tyr     1650              #   1655               #  1660 atc aaa cat gtt gtt gat aat aat tca cat at #t atc tat tca ggc cag     5040 Ile Lys His Val Val Asp Asn Asn Ser His Il #e Ile Tyr Ser Gly Gln 1665                1670 #                1675  #               1680 cta aca gat aca aat ata aac atc aca tta tt #t att cct ctt gat gat     5088 Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Ph #e Ile Pro Leu Asp Asp                 1685  #               1690   #              1695 gtc cca ttg aat caa gat tat cac gcc aag gt #t tat atg acc ttc aag     5136 Val Pro Leu Asn Gln Asp Tyr His Ala Lys Va #l Tyr Met Thr Phe Lys             1700      #           1705       #          1710 aaa tca cca tca gat ggt acc tgg tgg ggc cc #t cac ttt gtt aga gat     5184 Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pr #o His Phe Val Arg Asp         1715          #       1720           #      1725 gat aaa gga ata gta aca ata aac cct aaa tc #c att ttg acc cat ttt     5232 Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Se #r Ile Leu Thr His Phe     1730              #   1735               #  1740 gag agc gtc aat gtc ctg aat aat att agt ag #c gaa cca atg gat ttc     5280 Glu Ser Val Asn Val Leu Asn Asn Ile Ser Se #r Glu Pro Met Asp Phe 1745                1750 #                1755  #               1760 agc ggc gct aac agc ctc tat ttc tgg gaa ct #g ttc tac tat acc ccg     5328 Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Le #u Phe Tyr Tyr Thr Pro                 1765  #               1770   #              1775 atg ctg gtt gct caa cgt ttg ctg cat gaa ca #g aac ttc gat gaa gcc     5376 Met Leu Val Ala Gln Arg Leu Leu His Glu Gl #n Asn Phe Asp Glu Ala             1780      #           1785       #          1790 aac cgt tgg ctg aaa tat gtc tgg agt cca tc #c ggt tat att gtc cac     5424 Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Se #r Gly Tyr Ile Val His         1795          #       1800           #      1805 ggc cag att cag aac tac cag tgg aac gtc cg #c ccg tta ctg gaa gac     5472 Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Ar #g Pro Leu Leu Glu Asp     1810              #   1815               #  1820 acc agt tgg aac agt gat cct ttg gat tcc gt #c gat cct gac gcg gta     5520 Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Va #l Asp Pro Asp Ala Val 1825                1830 #                1835  #               1840 gca cag cac gat cca atg cac tac aaa gtt tc #a act ttt atg cgt acc     5568 Ala Gln His Asp Pro Met His Tyr Lys Val Se #r Thr Phe Met Arg Thr                 1845  #               1850   #              1855 ttg gat cta ttg ata gca cgc ggc gac cat gc #t tat cgc caa ctg gaa     5616 Leu Asp Leu Leu Ile Ala Arg Gly Asp His Al #a Tyr Arg Gln Leu Glu             1860      #           1865       #          1870 cga gat aca ctc aac gaa gcg aag atg tgg ta #t atg caa gcg ctg cat     5664 Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Ty #r Met Gln Ala Leu His         1875          #       1880           #      1885 cta tta ggt gac aaa cct tat cta ccg ctg ag #t acg aca tgg agt gat     5712 Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Se #r Thr Thr Trp Ser Asp     1890              #   1895               #  1900 cca cga cta gac aga gcc gcg gat atc act ac #c caa aat gct cac gac     5760 Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Th #r Gln Asn Ala His Asp 1905                1910 #                1915  #               1920 agc gca ata gtc gct ctg cgg cag aat ata cc #t aca ccg gca cct tta     5808 Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pr #o Thr Pro Ala Pro Leu                 1925  #               1930   #              1935 tca ttg cgc agc gct aat acc ctg act gat ct #c ttc ctg ccg caa atc     5856 Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Le #u Phe Leu Pro Gln Ile             1940      #           1945       #          1950 aat gaa gtg atg atg aat tac tgg cag aca tt #a gct cag aga gta tac     5904 Asn Glu Val Met Met Asn Tyr Trp Gln Thr Le #u Ala Gln Arg Val Tyr         1955          #       1960           #      1965 aat ctg cgt cat aac ctc tct atc gac ggc ca #g ccg tta tat ctg cca     5952 Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gl #n Pro Leu Tyr Leu Pro     1970              #   1975               #  1980 atc tat gcc aca ccg gcc gat ccg aaa gcg tt #a ctc agc gcc gcc gtt     6000 Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Le #u Leu Ser Ala Ala Val 1985                1990 #                1995  #               2000 gcc act tct caa ggt gga ggc aag cta ccg ga #a tca ttt atg tcc ctg     6048 Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Gl #u Ser Phe Met Ser Leu                 2005  #               2010   #              2015 tgg cgt ttc ccg cac atg ctg gaa aat gcg cg #c ggc atg gtt agc cag     6096 Trp Arg Phe Pro His Met Leu Glu Asn Ala Ar #g Gly Met Val Ser Gln             2020      #           2025       #          2030 ctc acc cag ttc ggc tcc acg tta caa aat at #t atc gaa cgt cag gac     6144 Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Il #e Ile Glu Arg Gln Asp         2035          #       2040           #      2045 gcg gaa gcg ctc aat gcg tta tta caa aat ca #g gcc gcc gag ctg ata     6192 Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gl #n Ala Ala Glu Leu Ile     2050              #   2055               #  2060 ttg act aac ctg agc att cag gac aaa acc at #t gaa gaa ttg gat gcc     6240 Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Il #e Glu Glu Leu Asp Ala 2065                2070 #                2075  #               2080 gag aaa acg gtg ttg gaa aaa tcc aaa gcg gg #a gca caa tcg cgc ttt     6288 Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gl #y Ala Gln Ser Arg Phe                 2085  #               2090   #              2095 gat agc tac ggc aaa ctg tac gat gag aat at #c aac gcc ggt gaa aac     6336 Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Il #e Asn Ala Gly Glu Asn             2100      #           2105       #          2110 caa gcc atg acg cta cga gcg tcc gcc gcc gg #g ctt acc acg gca gtt     6384 Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gl #y Leu Thr Thr Ala Val         2115          #       2120           #      2125 cag gca tcc cgt ctg gcc ggt gcg gcg gct ga #t ctg gtg cct aac atc     6432 Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala As #p Leu Val Pro Asn Ile     2130              #   2135               #  2140 ttc ggc ttt gcc ggt ggc ggc agc cgt tgg gg #g gct atc gct gag gcg     6480 Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gl #y Ala Ile Ala Glu Ala 2145                2150 #                2155  #               2160 aca ggt tat gtg atg gaa ttc tcc gcg aat gt #t atg aac acc gaa gcg     6528 Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Va #l Met Asn Thr Glu Ala                 2165  #               2170   #              2175 gat aaa att agc caa tct gaa acc tac cgt cg #t cgc cgt cag gag tgg     6576 Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Ar #g Arg Arg Gln Glu Trp             2180      #           2185       #          2190 gag atc cag cgg aat aat gcc gaa gcg gaa tt #g aag caa atc gat gct     6624 Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Le #u Lys Gln Ile Asp Ala         2195          #       2200           #      2205 cag ctc aaa tca ctc gct gta cgc cgc gaa gc #c gcc gta ttg cag aaa     6672 Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Al #a Ala Val Leu Gln Lys     2210              #   2215               #  2220 acc agt ctg aaa acc caa caa gaa cag acc ca #a tct caa ttg gcc ttc     6720 Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gl #n Ser Gln Leu Ala Phe 2225                2230 #                2235  #               2240 ctg caa cgt aag ttc agc aat cag gcg tta ta #c aac tgg ctg cgt ggt     6768 Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Ty #r Asn Trp Leu Arg Gly                 2245  #               2250   #              2255 cga ctg gcg gcg att tac ttc cag ttc tac ga #t ttg gcc gtc gcg cgt     6816 Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr As #p Leu Ala Val Ala Arg             2260      #           2265       #          2270 tgc ctg atg gca gaa caa gct tac cgt tgg ga #a ctc aat gat gac tct     6864 Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Gl #u Leu Asn Asp Asp Ser         2275          #       2280           #      2285 gcc cgc ttc att aaa ccg ggc gcc tgg cag gg #a acc tat gcc ggt ctg     6912 Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gl #y Thr Tyr Ala Gly Leu     2290              #   2295               #  2300 ctt gca ggt gaa acc ttg atg ctg agt ctg gc #a caa atg gaa gac gct     6960 Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Al #a Gln Met Glu Asp Ala 2305                2310 #                2315  #               2320 cat ctg aaa cgc gat aaa cgc gca tta gag gt #t gaa cgc aca gta tcg     7008 His Leu Lys Arg Asp Lys Arg Ala Leu Glu Va #l Glu Arg Thr Val Ser                 2325  #               2330   #              2335 ctg gcc gaa gtt tat gca gga tta cca aaa ga #t aac ggt cca ttt tcc     7056 Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys As #p Asn Gly Pro Phe Ser             2340      #           2345       #          2350 ctg gct cag gaa att gac aag ctg gtg agt ca #a ggt tca ggc agt gcc     7104 Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gl #n Gly Ser Gly Ser Ala         2355          #       2360           #      2365 ggc agt ggt aat aat aat ttg gcg ttc ggc gc #c ggc acg gac act aaa     7152 Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Al #a Gly Thr Asp Thr Lys     2370              #   2375               #  2380 acc tct ttg cag gca tca gtt tca ttc gct ga #t ttg aaa att cgt gaa     7200 Thr Ser Leu Gln Ala Ser Val Ser Phe Ala As #p Leu Lys Ile Arg Glu 2385                2390 #                2395  #               2400 gat tac ccg gca tcg ctt ggc aaa att cga cg #t atc aaa cag atc agc     7248 Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Ar #g Ile Lys Gln Ile Ser                 2405  #               2410   #              2415 gtc act ttg ccc gcg cta ctg gga ccg tat ca #g gat gta cag gca ata     7296 Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gl #n Asp Val Gln Ala Ile             2420      #           2425       #          2430 ttg tct tac ggc gat aaa gcc gga tta gct aa #c ggc tgt gaa gcg ctg     7344 Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala As #n Gly Cys Glu Ala Leu         2435          #       2440           #      2445 gca gtt tct cac ggt atg aat gac agc ggc ca #a ttc cag ctc gat ttc     7392 Ala Val Ser His Gly Met Asn Asp Ser Gly Gl #n Phe Gln Leu Asp Phe     2450              #   2455               #  2460 aac gat ggc aaa ttc ctg cca ttc gaa ggc at #c gcc att gat caa ggc     7440 Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Il #e Ala Ile Asp Gln Gly 2465                2470 #                2475  #               2480 acg ctg aca ctg agc ttc cca aat gca tct at #g ccg gag aaa ggt aaa     7488 Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Me #t Pro Glu Lys Gly Lys                 2485  #               2490   #              2495 caa gcc act atg tta aaa acc ctg aac gat at #c att ttg cat att cgc     7536 Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Il #e Ile Leu His Ile Arg             2500      #           2505       #          2510 tac acc att aaa taa            #                   #                   #  7551 Tyr Thr Ile Lys         2515 <210> SEQ ID NO 2 <211> LENGTH: 7515 <212> TYPE: DNA <213> ORGANISM: Photorhabdus luminescens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(7512) <400> SEQUENCE: 2 atg caa aac tca tta tca agc act atc gat ac #t att tgt cag aaa ctg       48 Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Th #r Ile Cys Gln Lys Leu   1               5  #                 10  #                 15 caa tta act tgt ccg gcg gaa att gct ttg ta #t ccc ttt gat act ttc       96 Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Ty #r Pro Phe Asp Thr Phe              20      #             25      #             30 cgg gaa aaa act cgg gga atg gtt aat tgg gg #g gaa gca aaa cgg att      144 Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gl #y Glu Ala Lys Arg Ile          35          #         40          #         45 tat gaa att gca caa gcg gaa cag gat aga aa #c cta ctt cat gaa aaa      192 Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg As #n Leu Leu His Glu Lys      50              #     55              #     60 cgt att ttt gcc tat gct aat ccg ctg ctg aa #a aac gct gtt cgg ttg      240 Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Ly #s Asn Ala Val Arg Leu  65                  # 70                  # 75                  # 80 ggt acc cgg caa atg ttg ggt ttt ata caa gg #t tat agt gat ctg ttt      288 Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gl #y Tyr Ser Asp Leu Phe                  85  #                 90  #                 95 ggt aat cgt gct gat aac tat gcc gcg ccg gg #c tcg gtt gca tcg atg      336 Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gl #y Ser Val Ala Ser Met             100       #           105       #           110 ttc tca ccg gcg gct tat ttg acg gaa ttg ta #c cgt gaa gcc aaa aac      384 Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Ty #r Arg Glu Ala Lys Asn         115           #       120           #       125 ttg cat gac agc agc tca att tat tac cta ga #t aaa cgt cgc ccg gat      432 Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu As #p Lys Arg Arg Pro Asp     130               #   135               #   140 tta gca agc tta atg ctc agc cag aaa aat at #g gat gag gaa att tca      480 Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Me #t Asp Glu Glu Ile Ser 145                 1 #50                 1 #55                 1 #60 acg ctg gct ctc tct aat gaa ttg tgc ctt gc #c ggg atc gaa aca aaa      528 Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Al #a Gly Ile Glu Thr Lys                 165   #               170   #               175 aca gga aaa tca caa gat gaa gtg atg gat at #g ttg tca act tat cgt      576 Thr Gly Lys Ser Gln Asp Glu Val Met Asp Me #t Leu Ser Thr Tyr Arg             180       #           185       #           190 tta agt gga gag aca cct tat cat cac gct ta #t gaa act gtt cgt gaa      624 Leu Ser Gly Glu Thr Pro Tyr His His Ala Ty #r Glu Thr Val Arg Glu         195           #       200           #       205 atc gtt cat gaa cgt gat cca gga ttt cgt ca #t ttg tca cag gca ccc      672 Ile Val His Glu Arg Asp Pro Gly Phe Arg Hi #s Leu Ser Gln Ala Pro     210               #   215               #   220 att gtt gct gct aag ctc gat cct gtg act tt #g ttg ggt att agc tcc      720 Ile Val Ala Ala Lys Leu Asp Pro Val Thr Le #u Leu Gly Ile Ser Ser 225                 2 #30                 2 #35                 2 #40 cat att tcg cca gaa ctg tat aac ttg ctg at #t gag gag atc ccg gaa      768 His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Il #e Glu Glu Ile Pro Glu                 245   #               250   #               255 aaa gat gaa gcc gcg ctt gat acg ctt tat aa #a aca aac ttt ggc gat      816 Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Ly #s Thr Asn Phe Gly Asp             260       #           265       #           270 att act act gct cag tta atg tcc cca agt ta #t ctg gcc cgg tat tat      864 Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Ty #r Leu Ala Arg Tyr Tyr         275           #       280           #       285 ggc gtc tca ccg gaa gat att gcc tac gtg ac #g act tca tta tca cat      912 Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Th #r Thr Ser Leu Ser His     290               #   295               #   300 gtt gga tat agc agt gat att ctg gtt att cc #g ttg gtc gat ggt gtg      960 Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pr #o Leu Val Asp Gly Val 305                 3 #10                 3 #15                 3 #20 ggt aag atg gaa gta gtt cgt gtt acc cga ac #a cca tcg gat aat tat     1008 Gly Lys Met Glu Val Val Arg Val Thr Arg Th #r Pro Ser Asp Asn Tyr                 325   #               330   #               335 acc agt cag acg aat tat att gag ctg tat cc #a cag ggt ggc gac aat     1056 Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pr #o Gln Gly Gly Asp Asn             340       #           345       #           350 tat ttg atc aaa tac aat cta agc aat agt tt #t ggt ttg gat gat ttt     1104 Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Ph #e Gly Leu Asp Asp Phe         355           #       360           #       365 tat ctg caa tat aaa gat ggt tcc gct gat tg #g act gag att gcc cat     1152 Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Tr #p Thr Glu Ile Ala His     370               #   375               #   380 aat ccc tat cct gat atg gtc ata aat caa aa #g tat gaa tca cag gcg     1200 Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Ly #s Tyr Glu Ser Gln Ala 385                 3 #90                 3 #95                 4 #00 aca atc aaa cgt agt gac tct gac aat ata ct #c agt ata ggg tta caa     1248 Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Le #u Ser Ile Gly Leu Gln                 405   #               410   #               415 aga tgg cat agc ggt agt tat aat ttt gcc gc #c gcc aat ttt aaa att     1296 Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Al #a Ala Asn Phe Lys Ile             420       #           425       #           430 gac caa tac tcc ccg aaa gct ttc ctg ctt aa #a atg aat aag gct att     1344 Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Ly #s Met Asn Lys Ala Ile         435           #       440           #       445 cgg ttg ctc aaa gct acc ggc ctc tct ttt gc #t acg ttg gag cgt att     1392 Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Al #a Thr Leu Glu Arg Ile     450               #   455               #   460 gtt gat agt gtt aat agc acc aaa tcc atc ac #g gtt gag gta tta aac     1440 Val Asp Ser Val Asn Ser Thr Lys Ser Ile Th #r Val Glu Val Leu Asn 465                 4 #70                 4 #75                 4 #80 aag gtt tat cgg gta aaa ttc tat att gat cg #t tat ggc atc agt gaa     1488 Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Ar #g Tyr Gly Ile Ser Glu                 485   #               490   #               495 gag aca gcc gct att ttg gct aat att aat at #c tct cag caa gct gtt     1536 Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Il #e Ser Gln Gln Ala Val             500       #           505       #           510 ggc aat cag ctt agc cag ttt gag caa cta tt #t aat cac ccg ccg ctc     1584 Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Ph #e Asn His Pro Pro Leu         515           #       520           #       525 aat ggt att cgc tat gaa atc agt gag gac aa #c tcc aaa cat ctt cct     1632 Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp As #n Ser Lys His Leu Pro     530               #   535               #   540 aat cct gat ctg aac ctt aaa cca gac agt ac #c ggt gat gat caa cgc     1680 Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Th #r Gly Asp Asp Gln Arg 545                 5 #50                 5 #55                 5 #60 aag gcg gtt tta aaa cgc gcg ttt cag gtt aa #c gcc agt gag ttg tat     1728 Lys Ala Val Leu Lys Arg Ala Phe Gln Val As #n Ala Ser Glu Leu Tyr                 565   #               570   #               575 cag atg tta ttg atc act gat cgt aaa gaa ga #c ggt gtt atc aaa aat     1776 Gln Met Leu Leu Ile Thr Asp Arg Lys Glu As #p Gly Val Ile Lys Asn             580       #           585       #           590 aac tta gag aat ttg tct gat ctg tat ttg gt #t agt ttg ctg gcc cag     1824 Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Va #l Ser Leu Leu Ala Gln         595           #       600           #       605 att cat aac ctg act att gct gaa ttg aac at #t ttg ttg gtg att tgt     1872 Ile His Asn Leu Thr Ile Ala Glu Leu Asn Il #e Leu Leu Val Ile Cys     610               #   615               #   620 ggc tat ggc gac acc aac att tat cag att ac #c gac gat aat tta gcc     1920 Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Th #r Asp Asp Asn Leu Ala 625                 6 #30                 6 #35                 6 #40 aaa ata gtg gaa aca ttg ttg tgg atc act ca #a tgg ttg aag acc caa     1968 Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gl #n Trp Leu Lys Thr Gln                 645   #               650   #               655 aaa tgg aca gtt acc gac ctg ttt ctg atg ac #c acg gcc act tac agc     2016 Lys Trp Thr Val Thr Asp Leu Phe Leu Met Th #r Thr Ala Thr Tyr Ser             660       #           665       #           670 acc act tta acg cca gaa att agc aat ctg ac #g gct acg ttg tct tca     2064 Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Th #r Ala Thr Leu Ser Ser         675           #       680           #       685 act ttg cat ggc aaa gag agt ctg att ggg ga #a gat ctg aaa aga gca     2112 Thr Leu His Gly Lys Glu Ser Leu Ile Gly Gl #u Asp Leu Lys Arg Ala     690               #   695               #   700 atg gcg cct tgc ttc act tcg gct ttg cat tt #g act tct caa gaa gtt     2160 Met Ala Pro Cys Phe Thr Ser Ala Leu His Le #u Thr Ser Gln Glu Val 705                 7 #10                 7 #15                 7 #20 gcg tat gac ctg ctg ttg tgg ata gac cag at #t caa ccg gca caa ata     2208 Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Il #e Gln Pro Ala Gln Ile                 725   #               730   #               735 act gtt gat ggg ttt tgg gaa gaa gtg caa ac #a aca cca acc agc ttg     2256 Thr Val Asp Gly Phe Trp Glu Glu Val Gln Th #r Thr Pro Thr Ser Leu             740       #           745       #           750 aag gtg att acc ttt gct cag gtg ctg gca ca #a ttg agc ctg atc tat     2304 Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gl #n Leu Ser Leu Ile Tyr         755           #       760           #       765 cgt cgt att ggg tta agt gaa acg gaa ctg tc #a ctg atc gtg act caa     2352 Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Se #r Leu Ile Val Thr Gln     770               #   775               #   780 tct tct ctg cta gtg gca ggc aaa agc ata ct #g gat cac ggt ctg tta     2400 Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Le #u Asp His Gly Leu Leu 785                 7 #90                 7 #95                 8 #00 acc ctg atg gcc ttg gaa ggt ttt cat acc tg #g gtt aat ggc ttg ggg     2448 Thr Leu Met Ala Leu Glu Gly Phe His Thr Tr #p Val Asn Gly Leu Gly                 805   #               810   #               815 caa cat gcc tcc ttg ata ttg gcg gcg ttg aa #a gac gga gcc ttg aca     2496 Gln His Ala Ser Leu Ile Leu Ala Ala Leu Ly #s Asp Gly Ala Leu Thr             820       #           825       #           830 gtt acc gat gta gca caa gct atg aat aag ga #g gaa tct ctc cta caa     2544 Val Thr Asp Val Ala Gln Ala Met Asn Lys Gl #u Glu Ser Leu Leu Gln         835           #       840           #       845 atg gca gct aat cag gtg gag aag gat cta ac #a aaa ctg acc agt tgg     2592 Met Ala Ala Asn Gln Val Glu Lys Asp Leu Th #r Lys Leu Thr Ser Trp     850               #   855               #   860 aca cag att gac gct att ctg caa tgg tta ca #g atg tct tcg gcc ttg     2640 Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gl #n Met Ser Ser Ala Leu 865                 8 #70                 8 #75                 8 #80 gcg gtt tct cca ctg gat ctg gca ggg atg at #g gcc ctg aaa tat ggg     2688 Ala Val Ser Pro Leu Asp Leu Ala Gly Met Me #t Ala Leu Lys Tyr Gly                 885   #               890   #               895 ata gat cat aac tat gct gcc tgg caa gct gc #g gcg gct gcg ctg atg     2736 Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Al #a Ala Ala Ala Leu Met             900       #           905       #           910 gct gat cat gct aat cag gca cag aaa aaa ct #g gat gag acg ttc agt     2784 Ala Asp His Ala Asn Gln Ala Gln Lys Lys Le #u Asp Glu Thr Phe Ser         915           #       920           #       925 aag gca tta tgt aac tat tat att aat gct gt #t gtc gat agt gct gct     2832 Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Va #l Val Asp Ser Ala Ala     930               #   935               #   940 gga gta cgt gat cgt aac ggt tta tat acc ta #t ttg ctg att gat aat     2880 Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Ty #r Leu Leu Ile Asp Asn 945                 9 #50                 9 #55                 9 #60 cag gtt tct gcc gat gtg atc act tca cgt at #t gca gaa gct atc gcc     2928 Gln Val Ser Ala Asp Val Ile Thr Ser Arg Il #e Ala Glu Ala Ile Ala                 965   #               970   #               975 ggt att caa ctg tac gtt aac cgg gct tta aa #c cga gat gaa ggt cag     2976 Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu As #n Arg Asp Glu Gly Gln             980       #           985       #           990 ctt gca tcg gac gtt agt acc cgt cag ttc tt #c act gac tgg gaa cgt     3024 Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Ph #e Thr Asp Trp Glu Arg         995           #       1000           #      1005 tac aat aaa cgt tac agt act tgg gct ggt gt #c tct gaa ctg gtc tat     3072 Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Va #l Ser Glu Leu Val Tyr     1010              #   1015               #  1020 tat cca gaa aac tat gtt gat ccc act cag cg #c att ggg caa acc aaa     3120 Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Ar #g Ile Gly Gln Thr Lys 1025                1030 #                1035  #               1040 atg atg gat gcg ctg ttg caa tcc atc aac ca #g agc cag cta aat gcg     3168 Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gl #n Ser Gln Leu Asn Ala                 1045  #               1050   #              1055 gat acg gtg gaa gat gct ttc aaa act tat tt #g acc agc ttt gag cag     3216 Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Le #u Thr Ser Phe Glu Gln             1060      #           1065       #          1070 gta gca aat ctg aaa gta att agt gct tac ca #c gat aat gtg aat gtg     3264 Val Ala Asn Leu Lys Val Ile Ser Ala Tyr Hi #s Asp Asn Val Asn Val         1075          #       1080           #      1085 gat caa gga tta act tat ttt atc ggt atc ga #c caa gca gct ccg ggt     3312 Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile As #p Gln Ala Ala Pro Gly     1090              #   1095               #  1100 acg tat tac tgg cgt agt gtt gat cac agc aa #a tgt gaa aat ggc aag     3360 Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Ly #s Cys Glu Asn Gly Lys 1105                1110 #                1115  #               1120 ttt gcc gct aat gct tgg ggt gag tgg aat aa #a att acc tgt gct gtc     3408 Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Ly #s Ile Thr Cys Ala Val                 1125  #               1130   #              1135 aat cct tgg aaa aat atc atc cgt ccg gtt gt #t tat atg tcc cgc tta     3456 Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Va #l Tyr Met Ser Arg Leu             1140      #           1145       #          1150 tat ctg cta tgg ctg gag cag caa tca aag aa #a agt gat gat ggt aaa     3504 Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Ly #s Ser Asp Asp Gly Lys         1155          #       1160           #      1165 acc acg att tat caa tat aac tta aaa ctg gc #t cat att cgt tac gac     3552 Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Al #a His Ile Arg Tyr Asp     1170              #   1175               #  1180 ggt agt tgg aat aca cca ttt act ttt gat gt #g aca gaa aag gta aaa     3600 Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Va #l Thr Glu Lys Val Lys 1185                1190 #                1195  #               1200 aat tac acg tcg agt act gat gct gct gaa tc #t tta ggg ttg tat tgt     3648 Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Se #r Leu Gly Leu Tyr Cys                 1205  #               1210   #              1215 act ggt tat caa ggg gaa gac act cta tta gt #t atg ttc tat tcg atg     3696 Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Va #l Met Phe Tyr Ser Met             1220      #           1225       #          1230 cag agt agt tat agc tcc tat acc gat aat aa #t gcg ccg gtc act ggg     3744 Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn As #n Ala Pro Val Thr Gly         1235          #       1240           #      1245 cta tat att ttc gct gat atg tca tca gac aa #t atg acg aat gca caa     3792 Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp As #n Met Thr Asn Ala Gln     1250              #   1255               #  1260 gca act aac tat tgg aat aac agt tat ccg ca #a ttt gat act gtg atg     3840 Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gl #n Phe Asp Thr Val Met 1265                1270 #                1275  #               1280 gca gat ccg gat agc gac aat aaa aaa gtc at #a acc aga aga gtt aat     3888 Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Il #e Thr Arg Arg Val Asn                 1285  #               1290   #              1295 aac cgt tat gcg gag gat tat gaa att cct tc #c tct gtg aca agt aac     3936 Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Se #r Ser Val Thr Ser Asn             1300      #           1305       #          1310 agt aat tat tct tgg ggt gat cac agt tta ac #c atg ctt tat ggt ggt     3984 Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Th #r Met Leu Tyr Gly Gly         1315          #       1320           #      1325 agt gtt cct aat att act ttt gaa tcg gcg gc #a gaa gat tta agg cta     4032 Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Al #a Glu Asp Leu Arg Leu     1330              #   1335               #  1340 tct acc aat atg gca ttg agt att att cat aa #t gga tat gcg gga acc     4080 Ser Thr Asn Met Ala Leu Ser Ile Ile His As #n Gly Tyr Ala Gly Thr 1345                1350 #                1355  #               1360 cgc cgt ata caa tgt aat ctt atg aaa caa ta #c gct tca tta ggt gat     4128 Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Ty #r Ala Ser Leu Gly Asp                 1365  #               1370   #              1375 aaa ttt ata att tat gat tca tca ttt gat ga #t gca aac cgt ttt aat     4176 Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp As #p Ala Asn Arg Phe Asn             1380      #           1385       #          1390 ctg gtg cca ttg ttt aaa ttc gga aaa gac ga #g aac tca gat gat agt     4224 Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Gl #u Asn Ser Asp Asp Ser         1395          #       1400           #      1405 att tgt ata tat aat gaa aac cct tcc tct ga #a gat aag aag tgg tat     4272 Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Gl #u Asp Lys Lys Trp Tyr     1410              #   1415               #  1420 ttt tct tcg aaa gat gac aat aaa aca gcg ga #t tat aat ggt gga act     4320 Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala As #p Tyr Asn Gly Gly Thr 1425                1430 #                1435  #               1440 caa tgt ata gat gct gga acc agt aac aaa ga #t ttt tat tat aat ctc     4368 Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys As #p Phe Tyr Tyr Asn Leu                 1445  #               1450   #              1455 cag gag att gaa gta att agt gtt act ggt gg #g tat tgg tcg agt tat     4416 Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gl #y Tyr Trp Ser Ser Tyr             1460      #           1465       #          1470 aaa ata tcc aac ccg att aat atc aat acg gg #c att gat agt gct aaa     4464 Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gl #y Ile Asp Ser Ala Lys         1475          #       1480           #      1485 gta aaa gtc acc gta aaa gcg ggt ggt gac ga #t caa atc ttt act gct     4512 Val Lys Val Thr Val Lys Ala Gly Gly Asp As #p Gln Ile Phe Thr Ala     1490              #   1495               #  1500 gat aat agt acc tat gtt cct cag caa ccg gc #a ccc agt ttt gag gag     4560 Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Al #a Pro Ser Phe Glu Glu 1505                1510 #                1515  #               1520 atg att tat cag ttc aat aac ctg aca ata ga #t tgt aag aat tta aat     4608 Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile As #p Cys Lys Asn Leu Asn                 1525  #               1530   #              1535 ttc atc gac aat cag gca cat att gag att ga #t ttc acc gct acg gca     4656 Phe Ile Asp Asn Gln Ala His Ile Glu Ile As #p Phe Thr Ala Thr Ala             1540      #           1545       #          1550 caa gat ggc cga ttc ttg ggt gca gaa act tt #t att atc ccg gta act     4704 Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Ph #e Ile Ile Pro Val Thr         1555          #       1560           #      1565 aaa aaa gtt ctc ggt act gag aac gtg att gc #g tta tat agc gaa aat     4752 Lys Lys Val Leu Gly Thr Glu Asn Val Ile Al #a Leu Tyr Ser Glu Asn     1570              #   1575               #  1580 aac ggt gtt caa tat atg caa att ggc gca ta #t cgt acc cgt ttg aat     4800 Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Ty #r Arg Thr Arg Leu Asn 1585                1590 #                1595  #               1600 acg tta ttc gct caa cag ttg gtt agc cgt gc #t aat cgt ggc att gat     4848 Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Al #a Asn Arg Gly Ile Asp                 1605  #               1610   #              1615 gca gtg ctc agt atg gaa act cag aat att ca #g gaa ccg caa tta gga     4896 Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gl #n Glu Pro Gln Leu Gly             1620      #           1625       #          1630 gcg ggc aca tat gtg cag ctt gtg ttg gat aa #a tat gat gag tct att     4944 Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Ly #s Tyr Asp Glu Ser Ile         1635          #       1640           #      1645 cat ggc act aat aaa agc ttt gct att gaa ta #t gtt gat ata ttt aaa     4992 His Gly Thr Asn Lys Ser Phe Ala Ile Glu Ty #r Val Asp Ile Phe Lys     1650              #   1655               #  1660 gag aac gat agt ttt gtg att tat caa gga ga #a ctt agc gaa aca agt     5040 Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Gl #u Leu Ser Glu Thr Ser 1665                1670 #                1675  #               1680 caa act gtt gtg aaa gtt ttc tta tcc tat tt #t ata gag gcg act gga     5088 Gln Thr Val Val Lys Val Phe Leu Ser Tyr Ph #e Ile Glu Ala Thr Gly                 1685  #               1690   #              1695 aat aag aac cac tta tgg gta cgt gct aaa ta #c caa aag gaa acg act     5136 Asn Lys Asn His Leu Trp Val Arg Ala Lys Ty #r Gln Lys Glu Thr Thr             1700      #           1705       #          1710 gat aag atc ttg ttc gac cgt act gat gag aa #a gat ccg cac ggt tgg     5184 Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Ly #s Asp Pro His Gly Trp         1715          #       1720           #      1725 ttt ctc agc gac gat cac aag acc ttt agt gg #t ctc tct tcc gca cag     5232 Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gl #y Leu Ser Ser Ala Gln     1730              #   1735               #  1740 gca tta aag aac gac agt gaa ccg atg gat tt #c tct ggc gcc aat gct     5280 Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Ph #e Ser Gly Ala Asn Ala 1745                1750 #                1755  #               1760 ctc tat ttc tgg gaa ctg ttc tat tac acg cc #g atg atg atg gct cat     5328 Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pr #o Met Met Met Ala His                 1765  #               1770   #              1775 cgt ttg ttg cag gaa cag aat ttt gat gcg gc #g aac cat tgg ttc cgt     5376 Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Al #a Asn His Trp Phe Arg             1780      #           1785       #          1790 tat gtc tgg agt cca tcc ggt tat atc gtt ga #t ggt aaa att gct atc     5424 Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val As #p Gly Lys Ile Ala Ile         1795          #       1800           #      1805 tac cac tgg aac gtg cga ccg ctg gaa gaa ga #c acc agt tgg aat gca     5472 Tyr His Trp Asn Val Arg Pro Leu Glu Glu As #p Thr Ser Trp Asn Ala     1810              #   1815               #  1820 caa caa ctg gac tcc acc gat cca gat gct gt #a gcc caa gat gat ccg     5520 Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Va #l Ala Gln Asp Asp Pro 1825                1830 #                1835  #               1840 atg cac tac aag gtg gct acc ttt atg gcg ac #g ttg gat ctg cta atg     5568 Met His Tyr Lys Val Ala Thr Phe Met Ala Th #r Leu Asp Leu Leu Met                 1845  #               1850   #              1855 gcc cgt ggt gat gct gct tac cgc cag tta ga #g cgt gat acg ttg gct     5616 Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Gl #u Arg Asp Thr Leu Ala             1860      #           1865       #          1870 gaa gct aaa atg tgg tat aca cag gcg ctt aa #t ctg ttg ggt gat gag     5664 Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu As #n Leu Leu Gly Asp Glu         1875          #       1880           #      1885 cca caa gtg atg ctg agt acg act tgg gct aa #t cca aca ttg ggt aat     5712 Pro Gln Val Met Leu Ser Thr Thr Trp Ala As #n Pro Thr Leu Gly Asn     1890              #   1895               #  1900 gct gct tca aaa acc aca cag cag gtt cgt ca #g caa gtg ctt acc cag     5760 Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gl #n Gln Val Leu Thr Gln 1905                1910 #                1915  #               1920 ttg cgt ctc aat agc agg gta aaa acc ccg tt #g cta gga aca gcc aat     5808 Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Le #u Leu Gly Thr Ala Asn                 1925  #               1930   #              1935 tcc ctg acc gct tta ttc ctg ccg cag gaa aa #t agc aag ctc aaa ggc     5856 Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu As #n Ser Lys Leu Lys Gly             1940      #           1945       #          1950 tac tgg cgg aca ctg gcg cag cgt atg ttt aa #t tta cgt cat aat ctg     5904 Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe As #n Leu Arg His Asn Leu         1955          #       1960           #      1965 tcg att gac ggc cag ccg ctc tcc ttg ccg ct #g tat gct aaa ccg gct     5952 Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Le #u Tyr Ala Lys Pro Ala     1970              #   1975               #  1980 gat cca aaa gct tta ctg agt gcg gcg gtt tc #a gct tct caa ggg gga     6000 Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Se #r Ala Ser Gln Gly Gly 1985                1990 #                1995  #               2000 gcc gac ttg ccg aag gcg ccg ctg act att ca #c cgc ttc cct caa atg     6048 Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile Hi #s Arg Phe Pro Gln Met                 2005  #               2010   #              2015 cta gaa ggg gca cgg ggc ttg gtt aac cag ct #t ata cag ttc ggt agt     6096 Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Le #u Ile Gln Phe Gly Ser             2020      #           2025       #          2030 tca cta ttg ggg tac agt gag cgt cag gat gc #g gaa gct atg agt caa     6144 Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Al #a Glu Ala Met Ser Gln         2035          #       2040           #      2045 cta ctg caa acc caa gcc agc gag tta ata ct #g acc agt att cgt atg     6192 Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Le #u Thr Ser Ile Arg Met     2050              #   2055               #  2060 cag gat aac caa ttg gca gag ctg gat tcg ga #a aaa acc gcc ttg caa     6240 Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Gl #u Lys Thr Ala Leu Gln 2065                2070 #                2075  #               2080 gtc tct tta gct gga gtg caa caa cgg ttt ga #c agc tat agc caa ctg     6288 Val Ser Leu Ala Gly Val Gln Gln Arg Phe As #p Ser Tyr Ser Gln Leu                 2085  #               2090   #              2095 tat gag gag aac atc aac gca ggt gag cag cg #a gcg ctg gcg tta cgc     6336 Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Ar #g Ala Leu Ala Leu Arg             2100      #           2105       #          2110 tca gaa tct gct att gag tct cag gga gcg ca #g att tcc cgt atg gca     6384 Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gl #n Ile Ser Arg Met Ala         2115          #       2120           #      2125 ggc gcg ggt gtt gat atg gca cca aat atc tt #c ggc ctg gct gat ggc     6432 Gly Ala Gly Val Asp Met Ala Pro Asn Ile Ph #e Gly Leu Ala Asp Gly     2130              #   2135               #  2140 ggc atg cat tat ggt gct att gcc tat gcc at #c gct gac ggt att gag     6480 Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Il #e Ala Asp Gly Ile Glu 2145                2150 #                2155  #               2160 ttg agt gct tct gcc aag atg gtt gat gcg ga #g aaa gtt gct cag tcg     6528 Leu Ser Ala Ser Ala Lys Met Val Asp Ala Gl #u Lys Val Ala Gln Ser                 2165  #               2170   #              2175 gaa ata tat cgc cgt cgc cgt caa gaa tgg aa #a att cag cgt gac aac     6576 Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Ly #s Ile Gln Arg Asp Asn             2180      #           2185       #          2190 gca caa gcg gag att aac cag tta aac gcg ca #a ctg gaa tca ctg tct     6624 Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gl #n Leu Glu Ser Leu Ser         2195          #       2200           #      2205 att cgc cgt gaa gcc gct gaa atg caa aaa ga #g tac ctg aaa acc cag     6672 Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Gl #u Tyr Leu Lys Thr Gln     2210              #   2215               #  2220 caa gct cag gcg cag gca caa ctt act ttc tt #a aga agc aaa ttc agt     6720 Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Le #u Arg Ser Lys Phe Ser 2225                2230 #                2235  #               2240 aat caa gcg tta tat agt tgg tta cga ggg cg #t ttg tca ggt att tat     6768 Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Ar #g Leu Ser Gly Ile Tyr                 2245  #               2250   #              2255 ttc cag ttc tat gac ttg gcc gta tca cgt tg #c ctg atg gca gag caa     6816 Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cy #s Leu Met Ala Glu Gln             2260      #           2265       #          2270 tcc tat caa tgg gaa gct aat gat aat tcc at #t agc ttt gtc aaa ccg     6864 Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Il #e Ser Phe Val Lys Pro         2275          #       2280           #      2285 ggt gca tgg caa gga act tac gcc ggc tta tt #g tgt gga gaa gct ttg     6912 Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Le #u Cys Gly Glu Ala Leu     2290              #   2295               #  2300 ata caa aat ctg gca caa atg gaa gag gca ta #t ctg aaa tgg gaa tct     6960 Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Ty #r Leu Lys Trp Glu Ser 2305                2310 #                2315  #               2320 cgc gct ttg gaa gta gaa cgc acg gtt tca tt #g gca gtg gtt tat gat     7008 Arg Ala Leu Glu Val Glu Arg Thr Val Ser Le #u Ala Val Val Tyr Asp                 2325  #               2330   #              2335 tca ctg gaa ggt aat gat cgt ttt aat tta gc #g gaa caa ata cct gca     7056 Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Al #a Glu Gln Ile Pro Ala             2340      #           2345       #          2350 tta ttg gat aag ggg gag gga aca gca gga ac #t aaa gaa aat ggg tta     7104 Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Th #r Lys Glu Asn Gly Leu         2355          #       2360           #      2365 tca ttg gct aat gct atc ctg tca gct tcg gt #c aaa ttg tcc gac ttg     7152 Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Va #l Lys Leu Ser Asp Leu     2370              #   2375               #  2380 aaa ctg gga acg gat tat cca gac agt atc gt #t ggt agc aac aag gtt     7200 Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Va #l Gly Ser Asn Lys Val 2385                2390 #                2395  #               2400 cgt cgt att aag caa atc agt gtt tcg cta cc #t gca ttg gtt ggg cct     7248 Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pr #o Ala Leu Val Gly Pro                 2405  #               2410   #              2415 tat cag gat gtt cag gct atg ctc agc tat gg #t ggc agt act caa ttg     7296 Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gl #y Gly Ser Thr Gln Leu             2420      #           2425       #          2430 ccg aaa ggt tgt tca gcg ttg gct gtg tct ca #t ggt acc aat gat agt     7344 Pro Lys Gly Cys Ser Ala Leu Ala Val Ser Hi #s Gly Thr Asn Asp Ser         2435          #       2440           #      2445 ggt cag ttc cag ttg gat ttc aat gac ggc aa #a tac ctg cca ttt gaa     7392 Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Ly #s Tyr Leu Pro Phe Glu     2450              #   2455               #  2460 ggt att gct ctt gat gat cag ggt aca ctg aa #t ctt caa ttt ccg aat     7440 Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu As #n Leu Gln Phe Pro Asn 2465                2470 #                2475  #               2480 gct acc gac aag cag aaa gca ata ttg caa ac #t atg agc gat att att     7488 Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Th #r Met Ser Asp Ile Ile                 2485  #               2490   #              2495 ttg cat att cgt tat acc atc cgt taa     #                   #           7515 Leu His Ile Arg Tyr Thr Ile Arg             2500 <210> SEQ ID NO 3 <211> LENGTH: 7577 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (3)..(7553) <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial  #Sequence:hemicot tcdA <400> SEQUENCE: 3 cc atg gct aac gag tcc gtc aag gag atc cca # gac gtc ctc aag tcc        47    Met Ala Asn Glu Ser Val Lys Glu Ile  #Pro Asp Val Leu Lys Ser      1              #  5                 #  10                 #  15 caa tgc ggt ttc aac tgc ctc act gac atc tc #c cac agc tcc ttc aac       95 Gln Cys Gly Phe Asn Cys Leu Thr Asp Ile Se #r His Ser Ser Phe Asn                  20  #                 25  #                 30 gag ttc aga caa caa gtc tct gag cac ctc tc #c tgg tcc gag acc cat      143 Glu Phe Arg Gln Gln Val Ser Glu His Leu Se #r Trp Ser Glu Thr His              35      #             40      #             45 gac ctc tac cat gac gct cag caa gct cag aa #g gac aac agg ctc tac      191 Asp Leu Tyr His Asp Ala Gln Gln Ala Gln Ly #s Asp Asn Arg Leu Tyr          50          #         55          #         60 gag gct agg atc ctc aag agg gct aac cca ca #a ctc cag aac gct gtc      239 Glu Ala Arg Ile Leu Lys Arg Ala Asn Pro Gl #n Leu Gln Asn Ala Val      65              #     70              #     75 cac ctc gcc atc ttg gct cca aac gct gag tt #g att ggt tac aac aac      287 His Leu Ala Ile Leu Ala Pro Asn Ala Glu Le #u Ile Gly Tyr Asn Asn  80                  # 85                  # 90                  # 95 cag ttc tct ggc aga gct agc cag tac gtg gc #t cct ggt aca gtc tcc      335 Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Al #a Pro Gly Thr Val Ser                 100   #               105   #               110 tcc atg ttc agc cca gcc gct tac ctc act ga #g ttg tac cgc gag gct      383 Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Gl #u Leu Tyr Arg Glu Ala             115       #           120       #           125 agg aac ctt cat gct tct gac tcc gtc tac ta #c ttg gac aca cgc aga      431 Arg Asn Leu His Ala Ser Asp Ser Val Tyr Ty #r Leu Asp Thr Arg Arg         130           #       135           #       140 cca gac ctc aag agc atg gcc ctc agc caa ca #g aac atg gac att gag      479 Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gl #n Asn Met Asp Ile Glu     145               #   150               #   155 ttg tcc acc ctc tcc ttg agc aac gag ctt ct #c ttg gag tcc atc aag      527 Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Le #u Leu Glu Ser Ile Lys 160                 1 #65                 1 #70                 1 #75 act gag agc aag ttg gag aac tac acc aag gt #c atg gag atg ctc tcc      575 Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Va #l Met Glu Met Leu Ser                 180   #               185   #               190 acc ttc aga cca agc ggt gca act cca tac ca #t gat gcc tac gag aac      623 Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr Hi #s Asp Ala Tyr Glu Asn             195       #           200       #           205 gtc agg gag gtc atc caa ctt caa gac cct gg #t ctt gag caa ctc aac      671 Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gl #y Leu Glu Gln Leu Asn         210           #       215           #       220 gct tct cca gcc att gct ggt ttg atg cac ca #g gca tcc ttg ctc ggt      719 Ala Ser Pro Ala Ile Ala Gly Leu Met His Gl #n Ala Ser Leu Leu Gly     225               #   230               #   235 atc aac gcc tcc atc tct cct gag ttg ttc aa #c atc ttg act gag gag      767 Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe As #n Ile Leu Thr Glu Glu 240                 2 #45                 2 #50                 2 #55 atc act gag ggc aac gct gag gag ttg tac aa #g aag aac ttc ggc aac      815 Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Ly #s Lys Asn Phe Gly Asn                 260   #               265   #               270 att gag cca gcc tct ctt gca atg cct gag ta #c ctc aag agg tac tac      863 Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Ty #r Leu Lys Arg Tyr Tyr             275       #           280       #           285 aac ttg tct gat gag gag ctt tct caa ttc at #t ggc aag gct tcc aac      911 Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Il #e Gly Lys Ala Ser Asn         290           #       295           #       300 ttc ggt caa cag gag tac agc aac aac cag ct #c atc act cca gtt gtg      959 Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Le #u Ile Thr Pro Val Val     305               #   310               #   315 aac tcc tct gat ggc act gtg aag gtc tac cg #c atc aca cgt gag tac     1007 Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Ar #g Ile Thr Arg Glu Tyr 320                 3 #25                 3 #30                 3 #35 acc aca aac gcc tac caa atg gat gtt gag tt #g ttc cca ttc ggt ggt     1055 Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Le #u Phe Pro Phe Gly Gly                 340   #               345   #               350 gag aac tac aga ctt gac tac aag ttc aag aa #c ttc tac aac gcc tcc     1103 Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys As #n Phe Tyr Asn Ala Ser             355       #           360       #           365 tac ctc tcc atc aag ttg aac gac aag agg ga #g ctt gtc agg act gag     1151 Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Gl #u Leu Val Arg Thr Glu         370           #       375           #       380 ggt gct cct caa gtg aac att gag tac tct gc #c aac atc acc ctc aac     1199 Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser Al #a Asn Ile Thr Leu Asn     385               #   390               #   395 aca gct gac atc tct caa cca ttc gag att gg #t ttg acc aga gtc ctt     1247 Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gl #y Leu Thr Arg Val Leu 400                 4 #05                 4 #10                 4 #15 ccc tct ggc tcc tgg gcc tac gct gca gcc aa #g ttc act gtt gag gag     1295 Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Ly #s Phe Thr Val Glu Glu                 420   #               425   #               430 tac aac cag tac tct ttc ctc ttg aag ctc aa #c aag gca att cgt ctc     1343 Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu As #n Lys Ala Ile Arg Leu             435       #           440       #           445 agc aga gcc act gag ttg tct ccc acc atc tt #g gag ggc att gtg agg     1391 Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Le #u Glu Gly Ile Val Arg         450           #       455           #       460 tct gtc aac ctt caa ctt gac atc aac act ga #t gtg ctt ggc aag gtc     1439 Ser Val Asn Leu Gln Leu Asp Ile Asn Thr As #p Val Leu Gly Lys Val     465               #   470               #   475 ttc ctc acc aag tac tac atg caa cgc tac gc #c atc cat gct gag act     1487 Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Al #a Ile His Ala Glu Thr 480                 4 #85                 4 #90                 4 #95 gca ctc atc ctc tgc aac gca ccc atc tct ca #a cgc tcc tac gac aac     1535 Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gl #n Arg Ser Tyr Asp Asn                 500   #               505   #               510 cag cct tcc cag ttc gac agg ctc ttc aac ac #t cct ctc ttg aac ggc     1583 Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn Th #r Pro Leu Leu Asn Gly             515       #           520       #           525 cag tac ttc tcc act ggt gat gag gag att ga #c ctc aac tct ggc tcc     1631 Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile As #p Leu Asn Ser Gly Ser         530           #       535           #       540 aca ggt gac tgg aga aag acc atc ttg aag ag #g gcc ttc aac att gat     1679 Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys Ar #g Ala Phe Asn Ile Asp     545               #   550               #   555 gat gtc tct ctc ttc cgt ctc ttg aag atc ac #a gat cac gac aac aag     1727 Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Th #r Asp His Asp Asn Lys 560                 5 #65                 5 #70                 5 #75 gat ggc aag atc aag aac aac ttg aag aac ct #t tcc aac ctc tac att     1775 Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Le #u Ser Asn Leu Tyr Ile                 580   #               585   #               590 ggc aag ttg ctt gca gac atc cac caa ctc ac #c att gat gag ttg gac     1823 Gly Lys Leu Leu Ala Asp Ile His Gln Leu Th #r Ile Asp Glu Leu Asp             595       #           600       #           605 ctc ttg ctc att gca gtc ggt gag ggc aag ac #c aac ctc tct gca atc     1871 Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Th #r Asn Leu Ser Ala Ile         610           #       615           #       620 tct gac aag cag ttg gca acc ctc atc agg aa #g ttg aac acc atc acc     1919 Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Ly #s Leu Asn Thr Ile Thr     625               #   630               #   635 tcc tgg ctt cac acc cag aag tgg tct gtc tt #c caa ctc ttc atc atg     1967 Ser Trp Leu His Thr Gln Lys Trp Ser Val Ph #e Gln Leu Phe Ile Met 640                 6 #45                 6 #50                 6 #55 acc agc acc tcc tac aac aag acc ctc act cc #t gag atc aag aac ctc     2015 Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pr #o Glu Ile Lys Asn Leu                 660   #               665   #               670 ttg gac aca gtc tac cac ggt ctc caa ggc tt #c gac aag gac aag gct     2063 Leu Asp Thr Val Tyr His Gly Leu Gln Gly Ph #e Asp Lys Asp Lys Ala             675       #           680       #           685 gac ttg ctt cat gtc atg gct ccc tac att gc #a gcc acc ctc caa ctc     2111 Asp Leu Leu His Val Met Ala Pro Tyr Ile Al #a Ala Thr Leu Gln Leu         690           #       695           #       700 tcc tct gag aac gtg gct cac tct gtc ttg ct #c tgg gct gac aag ctc     2159 Ser Ser Glu Asn Val Ala His Ser Val Leu Le #u Trp Ala Asp Lys Leu     705               #   710               #   715 caa cct ggt gat ggt gcc atg act gct gag aa #g ttc tgg gac tgg ctc     2207 Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Ly #s Phe Trp Asp Trp Leu 720                 7 #25                 7 #30                 7 #35 aac acc aag tac aca cca ggc tcc tct gag gc #t gtt gag act caa gag     2255 Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Al #a Val Glu Thr Gln Glu                 740   #               745   #               750 cac att gtg caa tac tgc cag gct ctt gca ca #g ttg gag atg gtc tac     2303 His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gl #n Leu Glu Met Val Tyr             755       #           760       #           765 cac tcc act ggc atc aac gag aac gct ttc ag #a ctc ttc gtc acc aag     2351 His Ser Thr Gly Ile Asn Glu Asn Ala Phe Ar #g Leu Phe Val Thr Lys         770           #       775           #       780 cct gag atg ttc ggt gct gcc aca ggt gct gc #a cct gct cat gat gct     2399 Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Al #a Pro Ala His Asp Ala     785               #   790               #   795 ctc tcc ctc atc atg ttg acc agg ttc gct ga #c tgg gtc aac gct ctt     2447 Leu Ser Leu Ile Met Leu Thr Arg Phe Ala As #p Trp Val Asn Ala Leu 800                 8 #05                 8 #10                 8 #15 ggt gag aag gct tcc tct gtc ttg gct gcc tt #c gag gcc aac tcc ctc     2495 Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Ph #e Glu Ala Asn Ser Leu                 820   #               825   #               830 act gct gag caa ctt gct gat gcc atg aac ct #t gat gcc aac ctc ttg     2543 Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Le #u Asp Ala Asn Leu Leu             835       #           840       #           845 ctc caa gct tcc att caa gct cag aac cac ca #a cac ctc cca cct gtc     2591 Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gl #n His Leu Pro Pro Val         850           #       855           #       860 act cca gag aac gct ttc tcc tgc tgg acc tc #c atc aac acc atc ctc     2639 Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Se #r Ile Asn Thr Ile Leu     865               #   870               #   875 caa tgg gtc aac gtg gct cag caa ctc aac gt #g gct cca caa ggt gtc     2687 Gln Trp Val Asn Val Ala Gln Gln Leu Asn Va #l Ala Pro Gln Gly Val 880                 8 #85                 8 #90                 8 #95 tct gct ttg gtc ggt ctt gac tac atc cag tc #c atg aag gag aca cca     2735 Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Se #r Met Lys Glu Thr Pro                 900   #               905   #               910 acc tac gct caa tgg gag aac gca gct ggt gt #c ttg act gct ggt ctc     2783 Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Va #l Leu Thr Ala Gly Leu             915       #           920       #           925 aac tcc caa cag gcc aac acc ctc cat gct tt #c ttg gat gag tct cgc     2831 Asn Ser Gln Gln Ala Asn Thr Leu His Ala Ph #e Leu Asp Glu Ser Arg         930           #       935           #       940 tct gct gcc ctc tcc acc tac tac atc agg ca #a gtc gcc aag gca gct     2879 Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gl #n Val Ala Lys Ala Ala     945               #   950               #   955 gct gcc atc aag tct cgc gat gac ctc tac ca #a tac ctc ctc att gac     2927 Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gl #n Tyr Leu Leu Ile Asp 960                 9 #65                 9 #70                 9 #75 aac cag gtc tct gct gcc atc aag acc acc ag #g atc gct gag gcc atc     2975 Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Ar #g Ile Ala Glu Ala Ile                 980   #               985   #               990 gct tcc atc caa ctc tac gtc aac cgc gct ct #t gag aac gtt gag gag     3023 Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Le #u Glu Asn Val Glu Glu             995       #           1000       #          1005 aac gcc aac tct ggt gtc atc tct cgc caa tt #c ttc atc gac tgg gac     3071 Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Ph #e Phe Ile Asp Trp Asp         1010          #       1015           #      1020 aag tac aac aag agg tac tcc acc tgg gct gg #t gtc tct caa ctt gtc     3119 Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gl #y Val Ser Gln Leu Val     1025              #   1030               #  1035 tac tac cca gag aac tac att gac cca acc at #g agg att ggt cag acc     3167 Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Me #t Arg Ile Gly Gln Thr 1040                1045 #                1050  #               1055 aag atg atg gat gct ctc ttg caa tct gtc tc #c caa agc caa ctc aac     3215 Lys Met Met Asp Ala Leu Leu Gln Ser Val Se #r Gln Ser Gln Leu Asn                 1060  #               1065   #              1070 gct gac act gtg gag gat gcc ttc atg agc ta #c ctc acc tcc ttc gag     3263 Ala Asp Thr Val Glu Asp Ala Phe Met Ser Ty #r Leu Thr Ser Phe Glu             1075      #           1080       #          1085 caa gtt gcc aac ctc aag gtc atc tct gct ta #c cat gac aac atc aac     3311 Gln Val Ala Asn Leu Lys Val Ile Ser Ala Ty #r His Asp Asn Ile Asn         1090          #       1095           #      1100 aac gac caa ggt ctc acc tac ttc att ggt ct #c tct gag act gat gct     3359 Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Le #u Ser Glu Thr Asp Ala     1105              #   1110               #  1115 ggt gag tac tac tgg aga tcc gtg gac cac ag #c aag ttc aac gat ggc     3407 Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Se #r Lys Phe Asn Asp Gly 1120                1125 #                1130  #               1135 aag ttc gct gca aac gct tgg tct gag tgg ca #c aag att gac tgc cct     3455 Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp Hi #s Lys Ile Asp Cys Pro                 1140  #               1145   #              1150 atc aac cca tac aag tcc acc atc aga cct gt #c atc tac aag agc cgc     3503 Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Va #l Ile Tyr Lys Ser Arg             1155      #           1160       #          1165 ctc tac ttg ctc tgg ctt gag cag aag gag at #c acc aag caa act ggc     3551 Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Il #e Thr Lys Gln Thr Gly         1170          #       1175           #      1180 aac tcc aag gat ggt tac caa act gag act ga #c tac cgc tac gag ttg     3599 Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr As #p Tyr Arg Tyr Glu Leu     1185              #   1190               #  1195 aag ttg gct cac atc cgc tac gat ggt acc tg #g aac act cca atc acc     3647 Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Tr #p Asn Thr Pro Ile Thr 1200                1205 #                1210  #               1215 ttc gat gtc aac aag aag atc agc gag ttg aa #g ttg gag aag aac cgt     3695 Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Ly #s Leu Glu Lys Asn Arg                 1220  #               1225   #              1230 gct cct ggt ctc tac tgc gct ggt tac caa gg #t gag gac acc ctc ttg     3743 Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gl #y Glu Asp Thr Leu Leu             1235      #           1240       #          1245 gtc atg ttc tac aac cag caa gac acc ctt ga #c tcc tac aag aac gct     3791 Val Met Phe Tyr Asn Gln Gln Asp Thr Leu As #p Ser Tyr Lys Asn Ala         1250          #       1255           #      1260 tcc atg caa ggt ctc tac atc ttc gct gac at #g gct tcc aag gac atg     3839 Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Me #t Ala Ser Lys Asp Met     1265              #   1270               #  1275 act cca gag caa agc aac gtc tac cgt gac aa #c tcc tac caa cag ttc     3887 Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp As #n Ser Tyr Gln Gln Phe 1280                1285 #                1290  #               1295 gac acc aac aac gtc agg cgt gtc aac aac ag #a tac gct gag gac tac     3935 Asp Thr Asn Asn Val Arg Arg Val Asn Asn Ar #g Tyr Ala Glu Asp Tyr                 1300  #               1305   #              1310 gag atc cca agc tct gtc agc tct cgc aag ga #c tac ggc tgg ggt gac     3983 Glu Ile Pro Ser Ser Val Ser Ser Arg Lys As #p Tyr Gly Trp Gly Asp             1315      #           1320       #          1325 tac tac ctc agc atg gtg tac aac ggt gac at #c cca acc atc aac tac     4031 Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Il #e Pro Thr Ile Asn Tyr         1330          #       1335           #      1340 aag gct gcc tct tcc gac ctc aaa atc tac at #c agc cca aag ctc agg     4079 Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Il #e Ser Pro Lys Leu Arg     1345              #   1350               #  1355 atc atc cac aac ggc tac gag ggt cag aag ag #g aac cag tgc aac ttg     4127 Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Ar #g Asn Gln Cys Asn Leu 1360                1365 #                1370  #               1375 atg aac aag tac ggc aag ttg ggt gac aag tt #c att gtc tac acc tct     4175 Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Ph #e Ile Val Tyr Thr Ser                 1380  #               1385   #              1390 ctt ggt gtc aac cca aac aac agc tcc aac aa #g ctc atg ttc tac cca     4223 Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Ly #s Leu Met Phe Tyr Pro             1395      #           1400       #          1405 gtc tac caa tac tct ggc aac acc tct ggt ct #c aac cag ggt aga ctc     4271 Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Le #u Asn Gln Gly Arg Leu         1410          #       1415           #      1420 ttg ttc cac agg gac acc acc tac cca agc aa #g gtg gag gct tgg att     4319 Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Ly #s Val Glu Ala Trp Ile     1425              #   1430               #  1435 cct ggt gcc aag agg tcc ctc acc aac cag aa #c gct gcc att ggt gat     4367 Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln As #n Ala Ala Ile Gly Asp 1440                1445 #                1450  #               1455 gac tac gcc aca gac tcc ctc aac aag cct ga #t gac ctc aag cag tac     4415 Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro As #p Asp Leu Lys Gln Tyr                 1460  #               1465   #              1470 atc ttc atg act gac tcc aag ggc aca gcc ac #t gat gtc tct ggt cca     4463 Ile Phe Met Thr Asp Ser Lys Gly Thr Ala Th #r Asp Val Ser Gly Pro             1475      #           1480       #          1485 gtg gag atc aac act gca atc agc cca gcc aa #g gtc caa atc att gtc     4511 Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Ly #s Val Gln Ile Ile Val         1490          #       1495           #      1500 aag gct ggt ggc aag gag caa acc ttc aca gc #t gac aag gat gtc tcc     4559 Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Al #a Asp Lys Asp Val Ser     1505              #   1510               #  1515 atc cag cca agc cca tcc ttc gat gag atg aa #c tac caa ttc aac gct     4607 Ile Gln Pro Ser Pro Ser Phe Asp Glu Met As #n Tyr Gln Phe Asn Ala 1520                1525 #                1530  #               1535 ctt gag att gat ggt tct ggc ctc aac ttc at #c aac aac tct gct tcc     4655 Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Il #e Asn Asn Ser Ala Ser                 1540  #               1545   #              1550 att gat gtc acc ttc act gcc ttc gct gag ga #t ggc cgc aag ttg ggt     4703 Ile Asp Val Thr Phe Thr Ala Phe Ala Glu As #p Gly Arg Lys Leu Gly             1555      #           1560       #          1565 tac gag agc ttc tcc atc cca gtc acc ctt aa #g gtt tcc act gac aac     4751 Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Ly #s Val Ser Thr Asp Asn         1570          #       1575           #      1580 gca ctc acc ctt cat cac aac gag aac ggt gc #t cag tac atg caa tgg     4799 Ala Leu Thr Leu His His Asn Glu Asn Gly Al #a Gln Tyr Met Gln Trp     1585              #   1590               #  1595 caa agc tac cgc acc agg ttg aac acc ctc tt #c gca agg caa ctt gtg     4847 Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Ph #e Ala Arg Gln Leu Val 1600                1605 #                1610  #               1615 gcc cgt gcc acc aca ggc att gac acc atc ct #c agc atg gag acc cag     4895 Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Le #u Ser Met Glu Thr Gln                 1620  #               1625   #              1630 aac atc caa gag cca cag ttg ggc aag ggt tt #c tac gcc acc ttc gtc     4943 Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Ph #e Tyr Ala Thr Phe Val             1635      #           1640       #          1645 atc cca cct tac aac ctc agc act cat ggt ga #t gag agg tgg ttc aag     4991 Ile Pro Pro Tyr Asn Leu Ser Thr His Gly As #p Glu Arg Trp Phe Lys         1650          #       1655           #      1660 ctc tac atc aag cac gtg gtt gac aac aac tc #c cac atc atc tac tct     5039 Leu Tyr Ile Lys His Val Val Asp Asn Asn Se #r His Ile Ile Tyr Ser     1665              #   1670               #  1675 ggt caa ctc act gac acc aac atc aac atc ac #c ctc ttc atc cca ctt     5087 Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Th #r Leu Phe Ile Pro Leu 1680                1685 #                1690  #               1695 gac gat gtc cca ctc aac cag gac tac cat gc #c aag gtc tac atg acc     5135 Asp Asp Val Pro Leu Asn Gln Asp Tyr His Al #a Lys Val Tyr Met Thr                 1700  #               1705   #              1710 ttc aag aag tct cca tct gat ggc acc tgg tg #g ggt cca cac ttc gtc     5183 Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Tr #p Gly Pro His Phe Val             1715      #           1720       #          1725 cgt gat gac aag ggc atc gtc acc atc aac cc #a aag tcc atc ctc acc     5231 Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pr #o Lys Ser Ile Leu Thr         1730          #       1735           #      1740 cac ttc gag tct gtc aac gtt ctc aac aac at #c tcc tct gag cca atg     5279 His Phe Glu Ser Val Asn Val Leu Asn Asn Il #e Ser Ser Glu Pro Met     1745              #   1750               #  1755 gac ttc tct ggt gcc aac tcc ctc tac ttc tg #g gag ttg ttc tac tac     5327 Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Tr #p Glu Leu Phe Tyr Tyr 1760                1765 #                1770  #               1775 aca cca atg ctt gtg gct caa agg ttg ctc ca #t gag cag aac ttc gat     5375 Thr Pro Met Leu Val Ala Gln Arg Leu Leu Hi #s Glu Gln Asn Phe Asp                 1780  #               1785   #              1790 gag gcc aac agg tgg ctc aag tac gtc tgg ag #c cca tct ggt tac att     5423 Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Se #r Pro Ser Gly Tyr Ile             1795      #           1800       #          1805 gtg cat ggt caa atc cag aac tac caa tgg aa #c gtc agg cca ttg ctt     5471 Val His Gly Gln Ile Gln Asn Tyr Gln Trp As #n Val Arg Pro Leu Leu         1810          #       1815           #      1820 gag gac acc tcc tgg aac tct gac cca ctt ga #c tct gtg gac cct gat     5519 Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu As #p Ser Val Asp Pro Asp     1825              #   1830               #  1835 gct gtg gct caa cat gac cca atg cac tac aa #g gtc tcc acc ttc atg     5567 Ala Val Ala Gln His Asp Pro Met His Tyr Ly #s Val Ser Thr Phe Met 1840                1845 #                1850  #               1855 agg acc ttg gac ctc ttg att gcc aga ggt ga #c cat gct tac cgc caa     5615 Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly As #p His Ala Tyr Arg Gln                 1860  #               1865   #              1870 ttg gag agg gac acc ctc aac gag gca aag at #g tgg tac atg caa gct     5663 Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Me #t Trp Tyr Met Gln Ala             1875      #           1880       #          1885 ctc cac ctc ttg ggt gac aag cca tac ctc cc #a ctc agc acc act tgg     5711 Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pr #o Leu Ser Thr Thr Trp         1890          #       1895           #      1900 tcc gac cca agg ttg gac cgt gct gct gac at #c acc act cag aac gct     5759 Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp Il #e Thr Thr Gln Asn Ala     1905              #   1910               #  1915 cat gac tct gcc att gtt gct ctc agg cag aa #c atc cca act cct gct     5807 His Asp Ser Ala Ile Val Ala Leu Arg Gln As #n Ile Pro Thr Pro Ala 1920                1925 #                1930  #               1935 cca ctc tcc ctc aga tct gct aac acc ctc ac #t gac ttg ttc ctc cca     5855 Pro Leu Ser Leu Arg Ser Ala Asn Thr Leu Th #r Asp Leu Phe Leu Pro                 1940  #               1945   #              1950 cag atc aac gag gtc atg atg aac tac tgg ca #a acc ttg gct caa agg     5903 Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gl #n Thr Leu Ala Gln Arg             1955      #           1960       #          1965 gtc tac aac ctc aga cac aac ctc tcc att ga #t ggt caa cca ctc tac     5951 Val Tyr Asn Leu Arg His Asn Leu Ser Ile As #p Gly Gln Pro Leu Tyr         1970          #       1975           #      1980 ctc cca atc tac gcc aca cca gct gac cca aa #g gct ctt ctc tct gct     5999 Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Ly #s Ala Leu Leu Ser Ala     1985              #   1990               #  1995 gct gtg gct acc agc caa ggt ggt ggc aag ct #c cca gag tcc ttc atg     6047 Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Le #u Pro Glu Ser Phe Met 2000                2005 #                2010  #               2015 tcc ctc tgg agg ttc cca cac atg ttg gag aa #c gcc cgt ggc atg gtc     6095 Ser Leu Trp Arg Phe Pro His Met Leu Glu As #n Ala Arg Gly Met Val                 2020  #               2025   #              2030 tcc caa ctc acc cag ttc ggt tcc acc ctc ca #g aac atc att gag agg     6143 Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gl #n Asn Ile Ile Glu Arg             2035      #           2040       #          2045 caa gat gct gag gct ctc aac gct ttg ctc ca #g aac cag gca gct gag     6191 Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gl #n Asn Gln Ala Ala Glu         2050          #       2055           #      2060 ttg atc ctc acc aac ttg tcc atc caa gac aa #g acc att gag gag ctt     6239 Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Ly #s Thr Ile Glu Glu Leu     2065              #   2070               #  2075 gat gct gag aag aca gtc ctt gag aag agc aa #g gct ggt gcc caa tct     6287 Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Ly #s Ala Gly Ala Gln Ser 2080                2085 #                2090  #               2095 cgc ttc gac tcc tac ggc aag ctc tac gat ga #g aac atc aac gct ggt     6335 Arg Phe Asp Ser Tyr Gly Lys Leu Tyr Asp Gl #u Asn Ile Asn Ala Gly                 2100  #               2105   #              2110 gag aac cag gcc atg acc ctc agg gct tcc gc #a gct ggt ctc acc act     6383 Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Al #a Ala Gly Leu Thr Thr             2115      #           2120       #          2125 gct gtc caa gcc tct cgc ttg gct ggt gca gc #t gct gac ctc gtt cca     6431 Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Al #a Ala Asp Leu Val Pro         2130          #       2135           #      2140 aac atc ttc ggt ttc gct ggt ggt ggc tcc ag #a tgg ggt gcc att gct     6479 Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Ar #g Trp Gly Ala Ile Ala     2145              #   2150               #  2155 gag gct acc ggt tac gtc atg gag ttc tct gc #c aac gtc atg aac act     6527 Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Al #a Asn Val Met Asn Thr 2160                2165 #                2170  #               2175 gag gct gac aag atc agc caa tct gag acc ta #c aga agg cgc cgt caa     6575 Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Ty #r Arg Arg Arg Arg Gln                 2180  #               2185   #              2190 gag tgg gag atc caa agg aac aac gct gag gc #a gag ttg aag caa atc     6623 Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Al #a Glu Leu Lys Gln Ile             2195      #           2200       #          2205 gat gct caa ctc aag tcc ttg gct gtc aga ag #g gag gct gct gtc ctc     6671 Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Ar #g Glu Ala Ala Val Leu         2210          #       2215           #      2220 cag aag acc tcc ctc aag acc caa cag gag ca #a acc cag tcc cag ttg     6719 Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gl #n Thr Gln Ser Gln Leu     2225              #   2230               #  2235 gct ttc ctc caa agg aag ttc tcc aac cag gc #t ctc tac aac tgg ctc     6767 Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Al #a Leu Tyr Asn Trp Leu 2240                2245 #                2250  #               2255 aga ggc cgc ttg gct gcc atc tac ttc caa tt #c tac gac ctt gct gtg     6815 Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Ph #e Tyr Asp Leu Ala Val                 2260  #               2265   #              2270 gcc agg tgc ctc atg gct gag caa gcc tac cg #c tgg gag ttg aac gat     6863 Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Ar #g Trp Glu Leu Asn Asp             2275      #           2280       #          2285 gac tcc gcc agg ttc atc aag cca ggt gct tg #g caa ggc acc tac gct     6911 Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Tr #p Gln Gly Thr Tyr Ala         2290          #       2295           #      2300 ggt ctc ctt gct ggt gag acc ctc atg ctc tc #c ttg gct caa atg gag     6959 Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Se #r Leu Ala Gln Met Glu     2305              #   2310               #  2315 gat gct cac ctc aag agg gac aag agg gct tt #g gag gtg gag agg aca     7007 Asp Ala His Leu Lys Arg Asp Lys Arg Ala Le #u Glu Val Glu Arg Thr 2320                2325 #                2330  #               2335 gtc tcc ctt gct gag gtc tac gct ggt ctc cc #a aag gac aac ggt cca     7055 Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pr #o Lys Asp Asn Gly Pro                 2340  #               2345   #              2350 ttc tcc ctt gct caa gag att gac aag ttg gt #c agc caa ggt tct ggt     7103 Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Va #l Ser Gln Gly Ser Gly             2355      #           2360       #          2365 tct gct ggt tct ggt aac aac aac ttg gct tt #c ggc gct ggt act gac     7151 Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Ph #e Gly Ala Gly Thr Asp         2370          #       2375           #      2380 acc aag acc tcc ctc caa gcc tct gtc tcc tt #c gct gac ctc aag atc     7199 Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Ph #e Ala Asp Leu Lys Ile     2385              #   2390               #  2395 agg gag gac tac cca gct tcc ctt ggc aag at #c agg cgc atc aag caa     7247 Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Il #e Arg Arg Ile Lys Gln 2400                2405 #                2410  #               2415 atc tct gtc acc ctc cca gct ctc ttg ggt cc #a tac caa gat gtc caa     7295 Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pr #o Tyr Gln Asp Val Gln                 2420  #               2425   #              2430 gca atc ctc tcc tac ggt gac aag gct ggt tt #g gcg aac ggt tgc gag     7343 Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Le #u Ala Asn Gly Cys Glu             2435      #           2440       #          2445 gct ctt gct gtc tct cat ggc atg aac gac tc #t ggt caa ttc caa ctt     7391 Ala Leu Ala Val Ser His Gly Met Asn Asp Se #r Gly Gln Phe Gln Leu         2450          #       2455           #      2460 gac ttc aac gat ggc aag ttc ctc cca ttc ga #g ggc att gcc att gac     7439 Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Gl #u Gly Ile Ala Ile Asp     2465              #   2470               #  2475 caa ggc acc ctc acc ctc tcc ttc cca aac gc #t tcc atg cca gag aag     7487 Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Al #a Ser Met Pro Glu Lys 2480                2485 #                2490  #               2495 gga aag caa gcc acc atg ctc aag acc ctc aa #c gat atc atc ctc cac     7535 Gly Lys Gln Ala Thr Met Leu Lys Thr Leu As #n Asp Ile Ile Leu His                 2500  #               2505   #              2510 atc agg tac acc atc aag tgagctcgag aggcctgcgg cc #gc                   #7577 Ile Arg Tyr Thr Ile Lys             2515 <210> SEQ ID NO 4 <211> LENGTH: 7541 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (3)..(7517) <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial  #Sequence:hemicot tcbA <400> SEQUENCE: 4 cc atg gct cag aac tcc ctc agc tcc acc att # gac acc atc tgc cag        47    Met Ala Gln Asn Ser Leu Ser Ser Thr  #Ile Asp Thr Ile Cys Gln      1              #  5                 #  10                 #  15 aag ctt caa ctc acc tgc cca gct gag atc gc #c ctc tac cca ttc gac       95 Lys Leu Gln Leu Thr Cys Pro Ala Glu Ile Al #a Leu Tyr Pro Phe Asp                  20  #                 25  #                 30 acc ttc cgt gag aag acc aga ggc atg gtc aa #c tgg ggt gag gcc aag      143 Thr Phe Arg Glu Lys Thr Arg Gly Met Val As #n Trp Gly Glu Ala Lys              35      #             40      #             45 agg atc tac gag att gct caa gct gag caa ga #c agg aac ctc ctt cat      191 Arg Ile Tyr Glu Ile Ala Gln Ala Glu Gln As #p Arg Asn Leu Leu His          50          #         55          #         60 gag aag agg atc ttc gcc tac gct aac cca tt #g ctc aag aac gct gtc      239 Glu Lys Arg Ile Phe Ala Tyr Ala Asn Pro Le #u Leu Lys Asn Ala Val      65              #     70              #     75 agg ctt ggt acc agg caa atg ttg ggt ttc at #c caa ggt tac tct gac      287 Arg Leu Gly Thr Arg Gln Met Leu Gly Phe Il #e Gln Gly Tyr Ser Asp  80                  # 85                  # 90                  # 95 ttg ttc ggc aac agg gct gac aac tac gca gc #t cct ggt tct gtt gct      335 Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Al #a Pro Gly Ser Val Ala                 100   #               105   #               110 agc atg ttc agc cca gct gcc tac ctc act ga #g ttg tac cgt gag gcc      383 Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Gl #u Leu Tyr Arg Glu Ala             115       #           120       #           125 aag aac ctc cat gac agc tcc agc atc tac ta #c ctt gac aag agg cgc      431 Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Ty #r Leu Asp Lys Arg Arg         130           #       135           #       140 cca gac ctt gct tcc ttg atg ctc tcc cag aa #g aac atg gat gag gag      479 Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Ly #s Asn Met Asp Glu Glu     145               #   150               #   155 atc agc acc ttg gct ctc tcc aac gag ctt tg #c ttg gct ggc att gag      527 Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cy #s Leu Ala Gly Ile Glu 160                 1 #65                 1 #70                 1 #75 acc aag act ggc aag tcc caa gat gag gtc at #g gac atg ctc tcc acc      575 Thr Lys Thr Gly Lys Ser Gln Asp Glu Val Me #t Asp Met Leu Ser Thr                 180   #               185   #               190 tac cgc ctc tct ggt gag act cca tac cac ca #t gct tac gag act gtc      623 Tyr Arg Leu Ser Gly Glu Thr Pro Tyr His Hi #s Ala Tyr Glu Thr Val             195       #           200       #           205 agg gag att gtc cat gag agg gac cca ggt tt #c cgc cac ctc tcc caa      671 Arg Glu Ile Val His Glu Arg Asp Pro Gly Ph #e Arg His Leu Ser Gln         210           #       215           #       220 gct ccc att gtg gct gcc aag ttg gac cca gt #c acc ctc ttg ggc atc      719 Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Va #l Thr Leu Leu Gly Ile     225               #   230               #   235 tcc agc cac atc agc cca gag ttg tac aac ct #t ctc att gag gag atc      767 Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Le #u Leu Ile Glu Glu Ile 240                 2 #45                 2 #50                 2 #55 cca gag aag gat gag gca gct ttg gac acc ct #c tac aag acc aac ttc      815 Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Le #u Tyr Lys Thr Asn Phe                 260   #               265   #               270 ggt gac atc acc act gct caa ctc atg agc cc #a tcc tac ttg gcc agg      863 Gly Asp Ile Thr Thr Ala Gln Leu Met Ser Pr #o Ser Tyr Leu Ala Arg             275       #           280       #           285 tac tac ggt gtc tct cca gag gac att gct ta #c gtc acc aca agc ctc      911 Tyr Tyr Gly Val Ser Pro Glu Asp Ile Ala Ty #r Val Thr Thr Ser Leu         290           #       295           #       300 tcc cat gtg ggt tac tcc tct gac atc ctt gt #c atc cca ctc gtg gat      959 Ser His Val Gly Tyr Ser Ser Asp Ile Leu Va #l Ile Pro Leu Val Asp     305               #   310               #   315 ggt gtg ggc aag atg gag gtt gtc agg gtc ac #c agg act cca tct gac     1007 Gly Val Gly Lys Met Glu Val Val Arg Val Th #r Arg Thr Pro Ser Asp 320                 3 #25                 3 #30                 3 #35 aac tac acc tcc cag acc aac tac att gag tt #g tac cca caa ggt ggt     1055 Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Le #u Tyr Pro Gln Gly Gly                 340   #               345   #               350 gac aac tac ctc atc aag tac aac ctc tcc aa #c tct ttc ggt ttg gat     1103 Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser As #n Ser Phe Gly Leu Asp             355       #           360       #           365 gac ttc tac ctc cag tac aag gat ggt tct gc #t gac tgg act gag att     1151 Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Al #a Asp Trp Thr Glu Ile         370           #       375           #       380 gct cac aac cca tac cca gac atg gtc atc aa #c cag aag tac gag tcc     1199 Ala His Asn Pro Tyr Pro Asp Met Val Ile As #n Gln Lys Tyr Glu Ser     385               #   390               #   395 caa gcc acc atc aag aga tct gac tct gac aa #c atc ctc tcc att ggt     1247 Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp As #n Ile Leu Ser Ile Gly 400                 4 #05                 4 #10                 4 #15 ctc caa agg tgg cac tct ggt tcc tac aac tt #c gct gct gcc aac ttc     1295 Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Ph #e Ala Ala Ala Asn Phe                 420   #               425   #               430 aag att gac caa tac tct cca aag gct ttc ct #c ttg aag atg aac aag     1343 Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Le #u Leu Lys Met Asn Lys             435       #           440       #           445 gcc atc agg ctc ttg aag gcc act ggt ctc tc #c ttc gcc acc ctt gag     1391 Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Se #r Phe Ala Thr Leu Glu         450           #       455           #       460 agg att gtg gac tct gtc aac tcc acc aag tc #c atc act gtg gag gtc     1439 Arg Ile Val Asp Ser Val Asn Ser Thr Lys Se #r Ile Thr Val Glu Val     465               #   470               #   475 ctc aac aag gtc tac aga gtc aag ttc tac at #t gac cgc tac ggc atc     1487 Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Il #e Asp Arg Tyr Gly Ile 480                 4 #85                 4 #90                 4 #95 tct gag gag act gct gcc atc ctt gcc aac at #c aac atc tcc cag caa     1535 Ser Glu Glu Thr Ala Ala Ile Leu Ala Asn Il #e Asn Ile Ser Gln Gln                 500   #               505   #               510 gct gtc ggc aac cag ctc tcc caa ttc gag ca #a ctc ttc aac cac cct     1583 Ala Val Gly Asn Gln Leu Ser Gln Phe Glu Gl #n Leu Phe Asn His Pro             515       #           520       #           525 cca ctc aac ggc atc cgc tac gag atc agc ga #g gac aac tcc aag cac     1631 Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Gl #u Asp Asn Ser Lys His         530           #       535           #       540 ctc cca aac cca gac ctc aac ctc aag cca ga #c tcc act ggt gat gac     1679 Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro As #p Ser Thr Gly Asp Asp     545               #   550               #   555 caa agg aag gct gtc ctc aag agg gct ttc ca #a gtc aac gct tct gag     1727 Gln Arg Lys Ala Val Leu Lys Arg Ala Phe Gl #n Val Asn Ala Ser Glu 560                 5 #65                 5 #70                 5 #75 ctt tac caa atg ctc ttg atc act gac agg aa #g gag gat ggt gtc atc     1775 Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Ly #s Glu Asp Gly Val Ile                 580   #               585   #               590 aag aac aac ttg gag aac ctc tct gac ctc ta #c ctt gtc tcc ctc ttg     1823 Lys Asn Asn Leu Glu Asn Leu Ser Asp Leu Ty #r Leu Val Ser Leu Leu             595       #           600       #           605 gcc caa atc cac aac ttg acc att gct gag tt #g aac atc ctc ttg gtc     1871 Ala Gln Ile His Asn Leu Thr Ile Ala Glu Le #u Asn Ile Leu Leu Val         610           #       615           #       620 atc tgc ggt tac ggt gac acc aac atc tac ca #a atc act gac gac aac     1919 Ile Cys Gly Tyr Gly Asp Thr Asn Ile Tyr Gl #n Ile Thr Asp Asp Asn     625               #   630               #   635 ctt gcc aag att gtg gag acc ctc ttg tgg at #c acc caa tgg ctc aag     1967 Leu Ala Lys Ile Val Glu Thr Leu Leu Trp Il #e Thr Gln Trp Leu Lys 640                 6 #45                 6 #50                 6 #55 acc cag aag tgg act gtc aca gac ctc ttc ct #c atg acc act gcc acc     2015 Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Le #u Met Thr Thr Ala Thr                 660   #               665   #               670 tac tcc acc act ctc act cca gag att tcc aa #c ctc act gcc acc ctc     2063 Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser As #n Leu Thr Ala Thr Leu             675       #           680       #           685 agc tcc acc ctc cac ggc aag gag tcc ctc at #t ggt gag gac ctc aag     2111 Ser Ser Thr Leu His Gly Lys Glu Ser Leu Il #e Gly Glu Asp Leu Lys         690           #       695           #       700 agg gca atg gct cca tgc ttc acc tct gct ct #c cac ctc acc tcc caa     2159 Arg Ala Met Ala Pro Cys Phe Thr Ser Ala Le #u His Leu Thr Ser Gln     705               #   710               #   715 gag gtg gct tac gac ctc ctt ctc tgg att ga #c caa atc caa cca gct     2207 Glu Val Ala Tyr Asp Leu Leu Leu Trp Ile As #p Gln Ile Gln Pro Ala 720                 7 #25                 7 #30                 7 #35 caa atc act gtg gat ggt ttc tgg gag gag gt #c caa acc act cca acc     2255 Gln Ile Thr Val Asp Gly Phe Trp Glu Glu Va #l Gln Thr Thr Pro Thr                 740   #               745   #               750 tcc ctc aag gtc atc acc ttc gct caa gtc tt #g gct caa ctc tcc ctc     2303 Ser Leu Lys Val Ile Thr Phe Ala Gln Val Le #u Ala Gln Leu Ser Leu             755       #           760       #           765 atc tac aga agg att ggt ctc tct gag act ga #g ttg tcc ctc att gtc     2351 Ile Tyr Arg Arg Ile Gly Leu Ser Glu Thr Gl #u Leu Ser Leu Ile Val         770           #       775           #       780 acc caa tcc agc ctc ttg gtc gct ggc aag tc #c atc ctt gat cat ggt     2399 Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Se #r Ile Leu Asp His Gly     785               #   790               #   795 ctc ttg acc ctc atg gct ctt gag ggt ttc ca #c acc tgg gtc aac ggt     2447 Leu Leu Thr Leu Met Ala Leu Glu Gly Phe Hi #s Thr Trp Val Asn Gly 800                 8 #05                 8 #10                 8 #15 ttg ggt caa cat gct tcc ctc atc ttg gct gc #a ctc aag gat ggt gct     2495 Leu Gly Gln His Ala Ser Leu Ile Leu Ala Al #a Leu Lys Asp Gly Ala                 820   #               825   #               830 ctc acc gtc acc gat gtg gct caa gcc atg aa #c aag gag gag tcc ctc     2543 Leu Thr Val Thr Asp Val Ala Gln Ala Met As #n Lys Glu Glu Ser Leu             835       #           840       #           845 ttg caa atg gct gcc aac cag gtg gag aag ga #c ctc acc aag ctc acc     2591 Leu Gln Met Ala Ala Asn Gln Val Glu Lys As #p Leu Thr Lys Leu Thr         850           #       855           #       860 tcc tgg acc caa atc gat gcc atc ctc caa tg #g ctc caa atg tcc tct     2639 Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Tr #p Leu Gln Met Ser Ser     865               #   870               #   875 gct ctt gct gtc agc cca ttg gac ctt gct gg #c atg atg gct ctc aag     2687 Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gl #y Met Met Ala Leu Lys 880                 8 #85                 8 #90                 8 #95 tac ggc att gat cac aac tac gct gcc tgg ca #a gca gct gcc gct gcc     2735 Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gl #n Ala Ala Ala Ala Ala                 900   #               905   #               910 ctc atg gct gac cat gcc aac cag gct cag aa #g aag ttg gat gag acc     2783 Leu Met Ala Asp His Ala Asn Gln Ala Gln Ly #s Lys Leu Asp Glu Thr             915       #           920       #           925 ttc tcc aag gct ctc tgc aac tac tac atc aa #c gcc gtg gtt gac tct     2831 Phe Ser Lys Ala Leu Cys Asn Tyr Tyr Ile As #n Ala Val Val Asp Ser         930           #       935           #       940 gct gcc ggt gtc agg gac agg aac ggt ctc ta #c acc tac ctc ttg att     2879 Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Ty #r Thr Tyr Leu Leu Ile     945               #   950               #   955 gac aac cag gtc tct gct gat gtc atc acc tc #c aga att gct gag gcc     2927 Asp Asn Gln Val Ser Ala Asp Val Ile Thr Se #r Arg Ile Ala Glu Ala 960                 9 #65                 9 #70                 9 #75 att gct ggc atc caa ctc tac gtc aac agg gc #t ctc aac agg gat gag     2975 Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Al #a Leu Asn Arg Asp Glu                 980   #               985   #               990 ggt cag ttg gct tct gat gtc tcc acc agg ca #a ttc ttc acc gac tgg     3023 Gly Gln Leu Ala Ser Asp Val Ser Thr Arg Gl #n Phe Phe Thr Asp Trp             995       #           1000       #          1005 gag agg tac aac aag agg tac tcc acc tgg gc #t ggt gtc tct gag ttg     3071 Glu Arg Tyr Asn Lys Arg Tyr Ser Thr Trp Al #a Gly Val Ser Glu Leu         1010          #       1015           #      1020 gtc tac tac cca gag aac tac gtg gac cca ac #c caa agg att ggt cag     3119 Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Th #r Gln Arg Ile Gly Gln     1025              #   1030               #  1035 acc aag atg atg gat gct ttg ctc caa tcc at #c aac cag tcc caa ctc     3167 Thr Lys Met Met Asp Ala Leu Leu Gln Ser Il #e Asn Gln Ser Gln Leu 1040                1045 #                1050  #               1055 aac gct gac act gtg gag gat gct ttc aag ac #c tac ctc acc tcc ttc     3215 Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Th #r Tyr Leu Thr Ser Phe                 1060  #               1065   #              1070 gag caa gtg gcc aac ctc aag gtc atc tct gc #t tac cat gac aac gtc     3263 Glu Gln Val Ala Asn Leu Lys Val Ile Ser Al #a Tyr His Asp Asn Val             1075      #           1080       #          1085 aac gtg gac caa ggt ctc acc tac ttc att gg #c att gac caa gcc gct     3311 Asn Val Asp Gln Gly Leu Thr Tyr Phe Ile Gl #y Ile Asp Gln Ala Ala         1090          #       1095           #      1100 cct ggc acc tac tac tgg agg tct gtg gac ca #c tcc aag tgc gag aac     3359 Pro Gly Thr Tyr Tyr Trp Arg Ser Val Asp Hi #s Ser Lys Cys Glu Asn     1105              #   1110               #  1115 ggc aag ttc gct gcc aac gct tgg ggt gag tg #g aac aag atc acc tgc     3407 Gly Lys Phe Ala Ala Asn Ala Trp Gly Glu Tr #p Asn Lys Ile Thr Cys 1120                1125 #                1130  #               1135 gct gtc aac cct tgg aag aac atc atc agg cc #a gtg gtc tac atg tcc     3455 Ala Val Asn Pro Trp Lys Asn Ile Ile Arg Pr #o Val Val Tyr Met Ser                 1140  #               1145   #              1150 aga ctc tac ttg ctc tgg ctt gag caa cag tc #c aag aag tct gat gac     3503 Arg Leu Tyr Leu Leu Trp Leu Glu Gln Gln Se #r Lys Lys Ser Asp Asp             1155      #           1160       #          1165 ggc aag aca act atc tac cag tac aac ctc aa #g ttg gct cac atc cgc     3551 Gly Lys Thr Thr Ile Tyr Gln Tyr Asn Leu Ly #s Leu Ala His Ile Arg         1170          #       1175           #      1180 tac gat ggt tcc tgg aac act cca ttc acc tt #c gat gtc act gag aag     3599 Tyr Asp Gly Ser Trp Asn Thr Pro Phe Thr Ph #e Asp Val Thr Glu Lys     1185              #   1190               #  1195 gtc aag aac tac acc tcc agc act gat gca gc #t gag tcc ctt ggt ctc     3647 Val Lys Asn Tyr Thr Ser Ser Thr Asp Ala Al #a Glu Ser Leu Gly Leu 1200                1205 #                1210  #               1215 tac tgc act ggt tac caa ggt gag gac acc ct #c ttg gtc atg ttc tac     3695 Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Le #u Leu Val Met Phe Tyr                 1220  #               1225   #              1230 tcc atg caa tcc agc tac tcc agc tac act ga #c aac aac gct cca gtc     3743 Ser Met Gln Ser Ser Tyr Ser Ser Tyr Thr As #p Asn Asn Ala Pro Val             1235      #           1240       #          1245 act ggt ctc tac atc ttc gct gac atg tcc tc #t gac aac atg acc aac     3791 Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Se #r Asp Asn Met Thr Asn         1250          #       1255           #      1260 gct caa gcc acc aac tac tgg aac aac tcc ta #c cca caa ttc gac act     3839 Ala Gln Ala Thr Asn Tyr Trp Asn Asn Ser Ty #r Pro Gln Phe Asp Thr     1265              #   1270               #  1275 gtc atg gct gac cca gac tct gac aac aag aa #g gtc atc acc agg cgt     3887 Val Met Ala Asp Pro Asp Ser Asp Asn Lys Ly #s Val Ile Thr Arg Arg 1280                1285 #                1290  #               1295 gtc aac aac cgc tac gct gag gac tac gag at #c cca agc tct gtc acc     3935 Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Il #e Pro Ser Ser Val Thr                 1300  #               1305   #              1310 tcc aac agc aac tac tcc tgg ggt gac cac tc #c ctc acc atg ctc tac     3983 Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Se #r Leu Thr Met Leu Tyr             1315      #           1320       #          1325 ggt ggc tct gtc cca aac atc acc ttc gag tc #t gca gct gag gac ctc     4031 Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Se #r Ala Ala Glu Asp Leu         1330          #       1335           #      1340 agg ctc tcc acc aac atg gct ctc tcc atc at #t cac aac ggt tac gct     4079 Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Il #e His Asn Gly Tyr Ala     1345              #   1350               #  1355 ggc acc agg cgc atc caa tgc aac ctc atg aa #g caa tac gct tcc ctt     4127 Gly Thr Arg Arg Ile Gln Cys Asn Leu Met Ly #s Gln Tyr Ala Ser Leu 1360                1365 #                1370  #               1375 ggt gac aag ttc att atc tac gac tcc agc tt #c gat gac gcc aac agg     4175 Gly Asp Lys Phe Ile Ile Tyr Asp Ser Ser Ph #e Asp Asp Ala Asn Arg                 1380  #               1385   #              1390 ttc aac ttg gtc cca ctc ttc aag ttc ggc aa #g gat gag aac tct gat     4223 Phe Asn Leu Val Pro Leu Phe Lys Phe Gly Ly #s Asp Glu Asn Ser Asp             1395      #           1400       #          1405 gac tcc atc tgc atc tac aac gag aac cca ag #c tct gag gac aag aag     4271 Asp Ser Ile Cys Ile Tyr Asn Glu Asn Pro Se #r Ser Glu Asp Lys Lys         1410          #       1415           #      1420 tgg tac ttc agc tcc aag gac gac aac aag ac #t gct gac tac aac ggt     4319 Trp Tyr Phe Ser Ser Lys Asp Asp Asn Lys Th #r Ala Asp Tyr Asn Gly     1425              #   1430               #  1435 ggc acc caa tgc att gat gct ggc acc tcc aa #c aag gac ttc tac tac     4367 Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser As #n Lys Asp Phe Tyr Tyr 1440                1445 #                1450  #               1455 aac ctc caa gag att gag gtc atc tct gtc ac #t ggt ggc tac tgg tcc     4415 Asn Leu Gln Glu Ile Glu Val Ile Ser Val Th #r Gly Gly Tyr Trp Ser                 1460  #               1465   #              1470 agc tac aag atc agc aac ccc atc aac atc aa #c act ggc att gac tct     4463 Ser Tyr Lys Ile Ser Asn Pro Ile Asn Ile As #n Thr Gly Ile Asp Ser             1475      #           1480       #          1485 gcc aag gtc aag gtc act gtc aag gct ggt gg #c gat gac caa atc ttc     4511 Ala Lys Val Lys Val Thr Val Lys Ala Gly Gl #y Asp Asp Gln Ile Phe         1490          #       1495           #      1500 act gct gac aac tcc acc tac gtc cca cag ca #a cct gct cca tcc ttc     4559 Thr Ala Asp Asn Ser Thr Tyr Val Pro Gln Gl #n Pro Ala Pro Ser Phe     1505              #   1510               #  1515 gag gag atg atc tac caa ttc aac aac ctc ac #c att gac tgc aag aac     4607 Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Th #r Ile Asp Cys Lys Asn 1520                1525 #                1530  #               1535 ctc aac ttc att gac aac cag gct cac att ga #g att gac ttc act gcc     4655 Leu Asn Phe Ile Asp Asn Gln Ala His Ile Gl #u Ile Asp Phe Thr Ala                 1540  #               1545   #              1550 aca gct caa gat ggc cgc ttc ttg ggt gct ga #g acc ttc atc att cca     4703 Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Gl #u Thr Phe Ile Ile Pro             1555      #           1560       #          1565 gtc acc aag aag gtc ctt ggc act gag aac gt #c att gct ctc tac tct     4751 Val Thr Lys Lys Val Leu Gly Thr Glu Asn Va #l Ile Ala Leu Tyr Ser         1570          #       1575           #      1580 gag aac aac ggt gtc cag tac atg caa att gg #t gct tac aga acc agg     4799 Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gl #y Ala Tyr Arg Thr Arg     1585              #   1590               #  1595 ctc aac acc ctc ttc gct caa cag ttg gtc tc #c cgt gcc aac aga ggc     4847 Leu Asn Thr Leu Phe Ala Gln Gln Leu Val Se #r Arg Ala Asn Arg Gly 1600                1605 #                1610  #               1615 att gat gct gtc ctc agc atg gag act cag aa #c atc caa gag cca caa     4895 Ile Asp Ala Val Leu Ser Met Glu Thr Gln As #n Ile Gln Glu Pro Gln                 1620  #               1625   #              1630 ctt ggt gct ggc acc tac gtc caa ctt gtc tt #g gac aag tac gat gag     4943 Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Le #u Asp Lys Tyr Asp Glu             1635      #           1640       #          1645 tcc att cat ggc acc aac aag tcc ttc gcc at #t gag tac gtg gac atc     4991 Ser Ile His Gly Thr Asn Lys Ser Phe Ala Il #e Glu Tyr Val Asp Ile         1650          #       1655           #      1660 ttc aag gag aac gac tcc ttc gtc atc tac ca #a ggt gag ttg tct gag     5039 Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gl #n Gly Glu Leu Ser Glu     1665              #   1670               #  1675 acc tcc caa act gtg gtc aag gtc ttc ctc tc #c tac ttc att gag gcc     5087 Thr Ser Gln Thr Val Val Lys Val Phe Leu Se #r Tyr Phe Ile Glu Ala 1680                1685 #                1690  #               1695 acc ggt aac aag aac cac ctc tgg gtc agg gc #c aag tac cag aag gag     5135 Thr Gly Asn Lys Asn His Leu Trp Val Arg Al #a Lys Tyr Gln Lys Glu                 1700  #               1705   #              1710 acc act gac aag atc ctc ttc gac agg act ga #t gag aag gac cca cat     5183 Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr As #p Glu Lys Asp Pro His             1715      #           1720       #          1725 ggt tgg ttc ctc tct gat gac cac aag acc tt #c tct ggt ctc agc tct     5231 Gly Trp Phe Leu Ser Asp Asp His Lys Thr Ph #e Ser Gly Leu Ser Ser         1730          #       1735           #      1740 gct caa gct ctc aag aac gac tct gag cca at #g gac ttc tct ggt gcc     5279 Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Me #t Asp Phe Ser Gly Ala     1745              #   1750               #  1755 aac gct ctc tac ttc tgg gag ttg ttc tac ta #c act cca atg atg atg     5327 Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Ty #r Thr Pro Met Met Met 1760                1765 #                1770  #               1775 gct cac agg ctc ctt caa gag cag aac ttc ga #t gct gcc aac cac tgg     5375 Ala His Arg Leu Leu Gln Glu Gln Asn Phe As #p Ala Ala Asn His Trp                 1780  #               1785   #              1790 ttc cgc tac gtc tgg agc cca tct ggt tac at #t gtg gat ggc aag att     5423 Phe Arg Tyr Val Trp Ser Pro Ser Gly Tyr Il #e Val Asp Gly Lys Ile             1795      #           1800       #          1805 gcc atc tac cac tgg aac gtc agg cca ttg ga #g gag gac acc tcc tgg     5471 Ala Ile Tyr His Trp Asn Val Arg Pro Leu Gl #u Glu Asp Thr Ser Trp         1810          #       1815           #      1820 aac gct cag caa ctt gac tcc act gac cca ga #t gct gtg gct caa gat     5519 Asn Ala Gln Gln Leu Asp Ser Thr Asp Pro As #p Ala Val Ala Gln Asp     1825              #   1830               #  1835 gac cca atg cac tac aag gtg gcc acc ttc at #g gcc acc ttg gac ctt     5567 Asp Pro Met His Tyr Lys Val Ala Thr Phe Me #t Ala Thr Leu Asp Leu 1840                1845 #                1850  #               1855 ctc atg gcc aga ggt gat gct gcc tac cgc ca #a ttg gag agg gac acc     5615 Leu Met Ala Arg Gly Asp Ala Ala Tyr Arg Gl #n Leu Glu Arg Asp Thr                 1860  #               1865   #              1870 ttg gct gag gcc aag atg tgg tac acc caa gc #t ctc aac ttg ctg ggt     5663 Leu Ala Glu Ala Lys Met Trp Tyr Thr Gln Al #a Leu Asn Leu Leu Gly             1875      #           1880       #          1885 gat gag cca caa gtc atg ctc tcc aca acc tg #g gcc aac cca acc ttg     5711 Asp Glu Pro Gln Val Met Leu Ser Thr Thr Tr #p Ala Asn Pro Thr Leu         1890          #       1895           #      1900 ggc aac gct gcc tcc aag acc aca caa cag gt #c agg caa cag gtc ctc     5759 Gly Asn Ala Ala Ser Lys Thr Thr Gln Gln Va #l Arg Gln Gln Val Leu     1905              #   1910               #  1915 acc caa ctc agg ctc aac tct aga gtc aag ac #t cca ctc ttg ggc act     5807 Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Th #r Pro Leu Leu Gly Thr 1920                1925 #                1930  #               1935 gcc aac tcc ctc act gct ctc ttc ctc cca ca #a gag aac tcc aaa ctt     5855 Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gl #n Glu Asn Ser Lys Leu                 1940  #               1945   #              1950 aag ggt tac tgg agg acc ctt gct caa cgc at #g ttc aac ctc agg cac     5903 Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Me #t Phe Asn Leu Arg His             1955      #           1960       #          1965 aac ctc tcc att gat ggt caa cca ctc tcc tt #g cca ctc tac gct aag     5951 Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Le #u Pro Leu Tyr Ala Lys         1970          #       1975           #      1980 cca gct gac cca aag gct ctc ctt tcc gct gc #t gtc tcc gca tcc caa     5999 Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Al #a Val Ser Ala Ser Gln     1985              #   1990               #  1995 ggt ggt gct gac ctc cca aag gct cca ctc ac #c atc cac agg ttc cca     6047 Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Th #r Ile His Arg Phe Pro 2000                2005 #                2010  #               2015 caa atg ttg gag ggt gcc cgt ggt ctt gtc aa #c cag ctc atc caa ttc     6095 Gln Met Leu Glu Gly Ala Arg Gly Leu Val As #n Gln Leu Ile Gln Phe                 2020  #               2025   #              2030 ggt tcc tct ctc ctt ggt tac tct gag agg ca #a gat gct gag gcc atg     6143 Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gl #n Asp Ala Glu Ala Met             2035      #           2040       #          2045 tcc caa ctc ttg caa acc cag gct tct gag tt #g atc ctc acc tcc atc     6191 Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Le #u Ile Leu Thr Ser Ile         2050          #       2055           #      2060 agg atg caa gac aac cag ctt gct gag ttg ga #c tct gag aag act gct     6239 Arg Met Gln Asp Asn Gln Leu Ala Glu Leu As #p Ser Glu Lys Thr Ala     2065              #   2070               #  2075 ctc caa gtc tcc ctt gct ggt gtc caa cag ag #g ttc gac agc tac tcc     6287 Leu Gln Val Ser Leu Ala Gly Val Gln Gln Ar #g Phe Asp Ser Tyr Ser 2080                2085 #                2090  #               2095 caa ctc tac gag gag aac atc aac gct ggt ga #g caa agg gct ttg gct     6335 Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Gl #u Gln Arg Ala Leu Ala                 2100  #               2105   #              2110 ctc agg tct gag tct gcc att gag tcc caa gg #t gct caa atc tcc cgc     6383 Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gl #y Ala Gln Ile Ser Arg             2115      #           2120       #          2125 atg gct ggt gct ggc gtg gac atg gct cca aa #c atc ttc ggt ctt gct     6431 Met Ala Gly Ala Gly Val Asp Met Ala Pro As #n Ile Phe Gly Leu Ala         2130          #       2135           #      2140 gat ggt ggc atg cac tac ggt gcc att gct ta #c gcc att gct gat ggc     6479 Asp Gly Gly Met His Tyr Gly Ala Ile Ala Ty #r Ala Ile Ala Asp Gly     2145              #   2150               #  2155 att gag ctt tct gct tct gcc aag atg gtt ga #t gct gag aag gtg gct     6527 Ile Glu Leu Ser Ala Ser Ala Lys Met Val As #p Ala Glu Lys Val Ala 2160                2165 #                2170  #               2175 caa tct gaa atc tac cgt cgc aga cgc caa ga #a tgg aag atc caa agg     6575 Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Gl #u Trp Lys Ile Gln Arg                 2180  #               2185   #              2190 gac aac gct caa gct gag atc aac cag ctc aa #c gct caa ctt gag tcc     6623 Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu As #n Ala Gln Leu Glu Ser             2195      #           2200       #          2205 ctc agc atc agg cgt gag gct gct gag atg ca #g aag gag tac ctc aag     6671 Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gl #n Lys Glu Tyr Leu Lys         2210          #       2215           #      2220 acc caa cag gct caa gct cag gct caa ctc ac #c ttc ctc agg tcc aag     6719 Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Th #r Phe Leu Arg Ser Lys     2225              #   2230               #  2235 ttc tcc aac cag gct ctc tac tcc tgg ctc ag #a ggc cgc ctc tct ggc     6767 Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Ar #g Gly Arg Leu Ser Gly 2240                2245 #                2250  #               2255 atc tac ttc caa ttc tac gac ttg gct gtc tc #c cgc tgc ctc atg gct     6815 Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Se #r Arg Cys Leu Met Ala                 2260  #               2265   #              2270 gag caa tcc tac caa tgg gag gcc aac gac aa #c agc atc tcc ttc gtc     6863 Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp As #n Ser Ile Ser Phe Val             2275      #           2280       #          2285 aag cca ggt gct tgg caa ggc acc tac gct gg #t ctc ctt tgc ggt gag     6911 Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gl #y Leu Leu Cys Gly Glu         2290          #       2295           #      2300 gct ctc atc cag aac ttg gct caa atg gag ga #g gct tac ctc aag tgg     6959 Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Gl #u Ala Tyr Leu Lys Trp     2305              #   2310               #  2315 gag tcc aga gct ttg gag gta gag agg act gt #c tcc ctt gct gta gtc     7007 Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Va #l Ser Leu Ala Val Val 2320                2325 #                2330  #               2335 tac gac tcc ttg gag ggc aac gac agg ttc aa #c ctt gct gag caa atc     7055 Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe As #n Leu Ala Glu Gln Ile                 2340  #               2345   #              2350 cca gct ctc ttg gac aag ggt gag ggc act gc #t ggc acc aag gag aac     7103 Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Al #a Gly Thr Lys Glu Asn             2355      #           2360       #          2365 ggt ctc tcc ttg gcc aac gcc atc ctc tct gc #t tct gtc aag ctc tct     7151 Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Al #a Ser Val Lys Leu Ser         2370          #       2375           #      2380 gac ctc aag ttg ggt act gac tac cca gac tc #c att gtg ggt tcc aac     7199 Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Se #r Ile Val Gly Ser Asn     2385              #   2390               #  2395 aag gtc aga agg atc aag caa atc tct gtc tc #c ctc cca gct ttg gtg     7247 Lys Val Arg Arg Ile Lys Gln Ile Ser Val Se #r Leu Pro Ala Leu Val 2400                2405 #                2410  #               2415 ggt cca tac caa gat gtc caa gcc atg ctc tc #c tac ggt ggc tcc acc     7295 Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Se #r Tyr Gly Gly Ser Thr                 2420  #               2425   #              2430 caa ctc cca aag ggt tgc tct gct ttg gct gt #c tcc cac ggc acc aac     7343 Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Va #l Ser His Gly Thr Asn             2435      #           2440       #          2445 gac tct ggt caa ttc caa ctt gac ttc aac ga #t ggc aag tac ctc cca     7391 Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn As #p Gly Lys Tyr Leu Pro         2450          #       2455           #      2460 ttc gaa ggc att gct ttg gat gac caa ggc ac #c ctc aac ctc caa ttc     7439 Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Th #r Leu Asn Leu Gln Phe     2465              #   2470               #  2475 cca aac gcc act gac aag cag aag gcc atc ct #c caa acc atg tct gac     7487 Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Le #u Gln Thr Met Ser Asp 2480                2485 #                2490  #               2495 atc atc ctc cac atc agg tac acc atc agg tg #agctcgag aggcctgcgg       7537 Ile Ile Leu His Ile Arg Tyr Thr Ile Arg                 2500  #               2505 ccgc                  #                   #                   #           7541 <210> SEQ ID NO 5 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial  #Sequence:hemicot       sequence encoding ER signal from  #15 kDa zein from Black       Mexican Sweet maize <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(63) <400> SEQUENCE: 5 atg gct aag atg gtc att gtg ctt gtg gtc tg #c ttg gct ctc tct gct       48 Met Ala Lys Met Val Ile Val Leu Val Val Cy #s Leu Ala Leu Ser Ala   1               5  #                 10  #                 15 gcc tgt gct tca gcc            #                   #                   #    63 Ala Cys Ala Ser Ala              20 <210> SEQ ID NO 6 <211> LENGTH: 7621 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial  #Sequence:hemicot tcdA       fused to the modified 15 kDa z #ein endoplasmic       reticulum signal peptide <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (4)..(7614) <220> FEATURE: unsure <221> NAME/KEY: (1) <223> OTHER INFORMATION: n can be a, t, g,  #or c <400> SEQUENCE: 6 ncc atg gct aag atg gtc att gtg ctt gtg gt #c tgc ttg gct ctc tct       48     Met Ala Lys Met Val Ile Val Leu  #Val Val Cys Leu Ala Leu Ser       1             #   5                #   10                #   15 gct gcc tgt gct tca gcc atg aac gag tcc gt #c aag gag atc cca gac       96 Ala Ala Cys Ala Ser Ala Met Asn Glu Ser Va #l Lys Glu Ile Pro Asp                  20  #                 25  #                 30 gtc ctc aag tcc caa tgc ggt ttc aac tgc ct #c act gac atc tcc cac      144 Val Leu Lys Ser Gln Cys Gly Phe Asn Cys Le #u Thr Asp Ile Ser His              35      #             40      #             45 agc tcc ttc aac gag ttc aga caa caa gtc tc #t gag cac ctc tcc tgg      192 Ser Ser Phe Asn Glu Phe Arg Gln Gln Val Se #r Glu His Leu Ser Trp          50          #         55          #         60 tcc gag acc cat gac ctc tac cat gac gct ca #g caa gct cag aag gac      240 Ser Glu Thr His Asp Leu Tyr His Asp Ala Gl #n Gln Ala Gln Lys Asp      65              #     70              #     75 aac agg ctc tac gag gct agg atc ctc aag ag #g gct aac cca caa ctc      288 Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Ar #g Ala Asn Pro Gln Leu  80                  # 85                  # 90                  # 95 cag aac gct gtc cac ctc gcc atc ttg gct cc #a aac gct gag ttg att      336 Gln Asn Ala Val His Leu Ala Ile Leu Ala Pr #o Asn Ala Glu Leu Ile                 100   #               105   #               110 ggt tac aac aac cag ttc tct ggc aga gct ag #c cag tac gtg gct cct      384 Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Se #r Gln Tyr Val Ala Pro             115       #           120       #           125 ggt aca gtc tcc tcc atg ttc agc cca gcc gc #t tac ctc act gag ttg      432 Gly Thr Val Ser Ser Met Phe Ser Pro Ala Al #a Tyr Leu Thr Glu Leu         130           #       135           #       140 tac cgc gag gct agg aac ctt cat gct tct ga #c tcc gtc tac tac ttg      480 Tyr Arg Glu Ala Arg Asn Leu His Ala Ser As #p Ser Val Tyr Tyr Leu     145               #   150               #   155 gac aca cgc aga cca gac ctc aag agc atg gc #c ctc agc caa cag aac      528 Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Al #a Leu Ser Gln Gln Asn 160                 1 #65                 1 #70                 1 #75 atg gac att gag ttg tcc acc ctc tcc ttg ag #c aac gag ctt ctc ttg      576 Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Se #r Asn Glu Leu Leu Leu                 180   #               185   #               190 gag tcc atc aag act gag agc aag ttg gag aa #c tac acc aag gtc atg      624 Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu As #n Tyr Thr Lys Val Met             195       #           200       #           205 gag atg ctc tcc acc ttc aga cca agc ggt gc #a act cca tac cat gat      672 Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Al #a Thr Pro Tyr His Asp         210           #       215           #       220 gcc tac gag aac gtc agg gag gtc atc caa ct #t caa gac cct ggt ctt      720 Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Le #u Gln Asp Pro Gly Leu     225               #   230               #   235 gag caa ctc aac gct tct cca gcc att gct gg #t ttg atg cac cag gca      768 Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gl #y Leu Met His Gln Ala 240                 2 #45                 2 #50                 2 #55 tcc ttg ctc ggt atc aac gcc tcc atc tct cc #t gag ttg ttc aac atc      816 Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pr #o Glu Leu Phe Asn Ile                 260   #               265   #               270 ttg act gag gag atc act gag ggc aac gct ga #g gag ttg tac aag aag      864 Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Gl #u Glu Leu Tyr Lys Lys             275       #           280       #           285 aac ttc ggc aac att gag cca gcc tct ctt gc #a atg cct gag tac ctc      912 Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Al #a Met Pro Glu Tyr Leu         290           #       295           #       300 aag agg tac tac aac ttg tct gat gag gag ct #t tct caa ttc att ggc      960 Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Le #u Ser Gln Phe Ile Gly     305               #   310               #   315 aag gct tcc aac ttc ggt caa cag gag tac ag #c aac aac cag ctc atc     1008 Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Se #r Asn Asn Gln Leu Ile 320                 3 #25                 3 #30                 3 #35 act cca gtt gtg aac tcc tct gat ggc act gt #g aag gtc tac cgc atc     1056 Thr Pro Val Val Asn Ser Ser Asp Gly Thr Va #l Lys Val Tyr Arg Ile                 340   #               345   #               350 aca cgt gag tac acc aca aac gcc tac caa at #g gat gtt gag ttg ttc     1104 Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Me #t Asp Val Glu Leu Phe             355       #           360       #           365 cca ttc ggt ggt gag aac tac aga ctt gac ta #c aag ttc aag aac ttc     1152 Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Ty #r Lys Phe Lys Asn Phe         370           #       375           #       380 tac aac gcc tcc tac ctc tcc atc aag ttg aa #c gac aag agg gag ctt     1200 Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu As #n Asp Lys Arg Glu Leu     385               #   390               #   395 gtc agg act gag ggt gct cct caa gtg aac at #t gag tac tct gcc aac     1248 Val Arg Thr Glu Gly Ala Pro Gln Val Asn Il #e Glu Tyr Ser Ala Asn 400                 4 #05                 4 #10                 4 #15 atc acc ctc aac aca gct gac atc tct caa cc #a ttc gag att ggt ttg     1296 Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pr #o Phe Glu Ile Gly Leu                 420   #               425   #               430 acc aga gtc ctt ccc tct ggc tcc tgg gcc ta #c gct gca gcc aag ttc     1344 Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Ty #r Ala Ala Ala Lys Phe             435       #           440       #           445 act gtt gag gag tac aac cag tac tct ttc ct #c ttg aag ctc aac aag     1392 Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Le #u Leu Lys Leu Asn Lys         450           #       455           #       460 gca att cgt ctc agc aga gcc act gag ttg tc #t ccc acc atc ttg gag     1440 Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Se #r Pro Thr Ile Leu Glu     465               #   470               #   475 ggc att gtg agg tct gtc aac ctt caa ctt ga #c atc aac act gat gtg     1488 Gly Ile Val Arg Ser Val Asn Leu Gln Leu As #p Ile Asn Thr Asp Val 480                 4 #85                 4 #90                 4 #95 ctt ggc aag gtc ttc ctc acc aag tac tac at #g caa cgc tac gcc atc     1536 Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Me #t Gln Arg Tyr Ala Ile                 500   #               505   #               510 cat gct gag act gca ctc atc ctc tgc aac gc #a ccc atc tct caa cgc     1584 His Ala Glu Thr Ala Leu Ile Leu Cys Asn Al #a Pro Ile Ser Gln Arg             515       #           520       #           525 tcc tac gac aac cag cct tcc cag ttc gac ag #g ctc ttc aac act cct     1632 Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Ar #g Leu Phe Asn Thr Pro         530           #       535           #       540 ctc ttg aac ggc cag tac ttc tcc act ggt ga #t gag gag att gac ctc     1680 Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly As #p Glu Glu Ile Asp Leu     545               #   550               #   555 aac tct ggc tcc aca ggt gac tgg aga aag ac #c atc ttg aag agg gcc     1728 Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Th #r Ile Leu Lys Arg Ala 560                 5 #65                 5 #70                 5 #75 ttc aac att gat gat gtc tct ctc ttc cgt ct #c ttg aag atc aca gat     1776 Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Le #u Leu Lys Ile Thr Asp                 580   #               585   #               590 cac gac aac aag gat ggc aag atc aag aac aa #c ttg aag aac ctt tcc     1824 His Asp Asn Lys Asp Gly Lys Ile Lys Asn As #n Leu Lys Asn Leu Ser             595       #           600       #           605 aac ctc tac att ggc aag ttg ctt gca gac at #c cac caa ctc acc att     1872 Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Il #e His Gln Leu Thr Ile         610           #       615           #       620 gat gag ttg gac ctc ttg ctc att gca gtc gg #t gag ggc aag acc aac     1920 Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gl #y Glu Gly Lys Thr Asn     625               #   630               #   635 ctc tct gca atc tct gac aag cag ttg gca ac #c ctc atc agg aag ttg     1968 Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Th #r Leu Ile Arg Lys Leu 640                 6 #45                 6 #50                 6 #55 aac acc atc acc tcc tgg ctt cac acc cag aa #g tgg tct gtc ttc caa     2016 Asn Thr Ile Thr Ser Trp Leu His Thr Gln Ly #s Trp Ser Val Phe Gln                 660   #               665   #               670 ctc ttc atc atg acc agc acc tcc tac aac aa #g acc ctc act cct gag     2064 Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Ly #s Thr Leu Thr Pro Glu             675       #           680       #           685 atc aag aac ctc ttg gac aca gtc tac cac gg #t ctc caa ggc ttc gac     2112 Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gl #y Leu Gln Gly Phe Asp         690           #       695           #       700 aag gac aag gct gac ttg ctt cat gtc atg gc #t ccc tac att gca gcc     2160 Lys Asp Lys Ala Asp Leu Leu His Val Met Al #a Pro Tyr Ile Ala Ala     705               #   710               #   715 acc ctc caa ctc tcc tct gag aac gtg gct ca #c tct gtc ttg ctc tgg     2208 Thr Leu Gln Leu Ser Ser Glu Asn Val Ala Hi #s Ser Val Leu Leu Trp 720                 7 #25                 7 #30                 7 #35 gct gac aag ctc caa cct ggt gat ggt gcc at #g act gct gag aag ttc     2256 Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Me #t Thr Ala Glu Lys Phe                 740   #               745   #               750 tgg gac tgg ctc aac acc aag tac aca cca gg #c tcc tct gag gct gtt     2304 Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gl #y Ser Ser Glu Ala Val             755       #           760       #           765 gag act caa gag cac att gtg caa tac tgc ca #g gct ctt gca cag ttg     2352 Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gl #n Ala Leu Ala Gln Leu         770           #       775           #       780 gag atg gtc tac cac tcc act ggc atc aac ga #g aac gct ttc aga ctc     2400 Glu Met Val Tyr His Ser Thr Gly Ile Asn Gl #u Asn Ala Phe Arg Leu     785               #   790               #   795 ttc gtc acc aag cct gag atg ttc ggt gct gc #c aca ggt gct gca cct     2448 Phe Val Thr Lys Pro Glu Met Phe Gly Ala Al #a Thr Gly Ala Ala Pro 800                 8 #05                 8 #10                 8 #15 gct cat gat gct ctc tcc ctc atc atg ttg ac #c agg ttc gct gac tgg     2496 Ala His Asp Ala Leu Ser Leu Ile Met Leu Th #r Arg Phe Ala Asp Trp                 820   #               825   #               830 gtc aac gct ctt ggt gag aag gct tcc tct gt #c ttg gct gcc ttc gag     2544 Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Va #l Leu Ala Ala Phe Glu             835       #           840       #           845 gcc aac tcc ctc act gct gag caa ctt gct ga #t gcc atg aac ctt gat     2592 Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala As #p Ala Met Asn Leu Asp         850           #       855           #       860 gcc aac ctc ttg ctc caa gct tcc att caa gc #t cag aac cac caa cac     2640 Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Al #a Gln Asn His Gln His     865               #   870               #   875 ctc cca cct gtc act cca gag aac gct ttc tc #c tgc tgg acc tcc atc     2688 Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Se #r Cys Trp Thr Ser Ile 880                 8 #85                 8 #90                 8 #95 aac acc atc ctc caa tgg gtc aac gtg gct ca #g caa ctc aac gtg gct     2736 Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gl #n Gln Leu Asn Val Ala                 900   #               905   #               910 cca caa ggt gtc tct gct ttg gtc ggt ctt ga #c tac atc cag tcc atg     2784 Pro Gln Gly Val Ser Ala Leu Val Gly Leu As #p Tyr Ile Gln Ser Met             915       #           920       #           925 aag gag aca cca acc tac gct caa tgg gag aa #c gca gct ggt gtc ttg     2832 Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu As #n Ala Ala Gly Val Leu         930           #       935           #       940 act gct ggt ctc aac tcc caa cag gcc aac ac #c ctc cat gct ttc ttg     2880 Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Th #r Leu His Ala Phe Leu     945               #   950               #   955 gat gag tct cgc tct gct gcc ctc tcc acc ta #c tac atc agg caa gtc     2928 Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Ty #r Tyr Ile Arg Gln Val 960                 9 #65                 9 #70                 9 #75 gcc aag gca gct gct gcc atc aag tct cgc ga #t gac ctc tac caa tac     2976 Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg As #p Asp Leu Tyr Gln Tyr                 980   #               985   #               990 ctc ctc att gac aac cag gtc tct gct gcc at #c aag acc acc agg atc     3024 Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Il #e Lys Thr Thr Arg Ile             995       #           1000       #          1005 gct gag gcc atc gct tcc atc caa ctc tac gt #c aac cgc gct ctt gag     3072 Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Va #l Asn Arg Ala Leu Glu         1010          #       1015           #      1020 aac gtt gag gag aac gcc aac tct ggt gtc at #c tct cgc caa ttc ttc     3120 Asn Val Glu Glu Asn Ala Asn Ser Gly Val Il #e Ser Arg Gln Phe Phe     1025              #   1030               #  1035 atc gac tgg gac aag tac aac aag agg tac tc #c acc tgg gct ggt gtc     3168 Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Se #r Thr Trp Ala Gly Val 1040                1045 #                1050  #               1055 tct caa ctt gtc tac tac cca gag aac tac at #t gac cca acc atg agg     3216 Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Il #e Asp Pro Thr Met Arg                 1060  #               1065   #              1070 att ggt cag acc aag atg atg gat gct ctc tt #g caa tct gtc tcc caa     3264 Ile Gly Gln Thr Lys Met Met Asp Ala Leu Le #u Gln Ser Val Ser Gln             1075      #           1080       #          1085 agc caa ctc aac gct gac act gtg gag gat gc #c ttc atg agc tac ctc     3312 Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Al #a Phe Met Ser Tyr Leu         1090          #       1095           #      1100 acc tcc ttc gag caa gtt gcc aac ctc aag gt #c atc tct gct tac cat     3360 Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Va #l Ile Ser Ala Tyr His     1105              #   1110               #  1115 gac aac atc aac aac gac caa ggt ctc acc ta #c ttc att ggt ctc tct     3408 Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Ty #r Phe Ile Gly Leu Ser 1120                1125 #                1130  #               1135 gag act gat gct ggt gag tac tac tgg aga tc #c gtg gac cac agc aag     3456 Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Se #r Val Asp His Ser Lys                 1140  #               1145   #              1150 ttc aac gat ggc aag ttc gct gca aac gct tg #g tct gag tgg cac aag     3504 Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Tr #p Ser Glu Trp His Lys             1155      #           1160       #          1165 att gac tgc cct atc aac cca tac aag tcc ac #c atc aga cct gtc atc     3552 Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Th #r Ile Arg Pro Val Ile         1170          #       1175           #      1180 tac aag agc cgc ctc tac ttg ctc tgg ctt ga #g cag aag gag atc acc     3600 Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Gl #u Gln Lys Glu Ile Thr     1185              #   1190               #  1195 aag caa act ggc aac tcc aag gat ggt tac ca #a act gag act gac tac     3648 Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gl #n Thr Glu Thr Asp Tyr 1200                1205 #                1210  #               1215 cgc tac gag ttg aag ttg gct cac atc cgc ta #c gat ggt acc tgg aac     3696 Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Ty #r Asp Gly Thr Trp Asn                 1220  #               1225   #              1230 act cca atc acc ttc gat gtc aac aag aag at #c agc gag ttg aag ttg     3744 Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Il #e Ser Glu Leu Lys Leu             1235      #           1240       #          1245 gag aag aac cgt gct cct ggt ctc tac tgc gc #t ggt tac caa ggt gag     3792 Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Al #a Gly Tyr Gln Gly Glu         1250          #       1255           #      1260 gac acc ctc ttg gtc atg ttc tac aac cag ca #a gac acc ctt gac tcc     3840 Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gl #n Asp Thr Leu Asp Ser     1265              #   1270               #  1275 tac aag aac gct tcc atg caa ggt ctc tac at #c ttc gct gac atg gct     3888 Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Il #e Phe Ala Asp Met Ala 1280                1285 #                1290  #               1295 tcc aag gac atg act cca gag caa agc aac gt #c tac cgt gac aac tcc     3936 Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Va #l Tyr Arg Asp Asn Ser                 1300  #               1305   #              1310 tac caa cag ttc gac acc aac aac gtc agg cg #t gtc aac aac aga tac     3984 Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Ar #g Val Asn Asn Arg Tyr             1315      #           1320       #          1325 gct gag gac tac gag atc cca agc tct gtc ag #c tct cgc aag gac tac     4032 Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Se #r Ser Arg Lys Asp Tyr         1330          #       1335           #      1340 ggc tgg ggt gac tac tac ctc agc atg gtg ta #c aac ggt gac atc cca     4080 Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Ty #r Asn Gly Asp Ile Pro     1345              #   1350               #  1355 acc atc aac tac aag gct gcc tct tcc gac ct #c aaa atc tac atc agc     4128 Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Le #u Lys Ile Tyr Ile Ser 1360                1365 #                1370  #               1375 cca aag ctc agg atc atc cac aac ggc tac ga #g ggt cag aag agg aac     4176 Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Gl #u Gly Gln Lys Arg Asn                 1380  #               1385   #              1390 cag tgc aac ttg atg aac aag tac ggc aag tt #g ggt gac aag ttc att     4224 Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Le #u Gly Asp Lys Phe Ile             1395      #           1400       #          1405 gtc tac acc tct ctt ggt gtc aac cca aac aa #c agc tcc aac aag ctc     4272 Val Tyr Thr Ser Leu Gly Val Asn Pro Asn As #n Ser Ser Asn Lys Leu         1410          #       1415           #      1420 atg ttc tac cca gtc tac caa tac tct ggc aa #c acc tct ggt ctc aac     4320 Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly As #n Thr Ser Gly Leu Asn     1425              #   1430               #  1435 cag ggt aga ctc ttg ttc cac agg gac acc ac #c tac cca agc aag gtg     4368 Gln Gly Arg Leu Leu Phe His Arg Asp Thr Th #r Tyr Pro Ser Lys Val 1440                1445 #                1450  #               1455 gag gct tgg att cct ggt gcc aag agg tcc ct #c acc aac cag aac gct     4416 Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Le #u Thr Asn Gln Asn Ala                 1460  #               1465   #              1470 gcc att ggt gat gac tac gcc aca gac tcc ct #c aac aag cct gat gac     4464 Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Le #u Asn Lys Pro Asp Asp             1475      #           1480       #          1485 ctc aag cag tac atc ttc atg act gac tcc aa #g ggc aca gcc act gat     4512 Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Ly #s Gly Thr Ala Thr Asp         1490          #       1495           #      1500 gtc tct ggt cca gtg gag atc aac act gca at #c agc cca gcc aag gtc     4560 Val Ser Gly Pro Val Glu Ile Asn Thr Ala Il #e Ser Pro Ala Lys Val     1505              #   1510               #  1515 caa atc att gtc aag gct ggt ggc aag gag ca #a acc ttc aca gct gac     4608 Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gl #n Thr Phe Thr Ala Asp 1520                1525 #                1530  #               1535 aag gat gtc tcc atc cag cca agc cca tcc tt #c gat gag atg aac tac     4656 Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Ph #e Asp Glu Met Asn Tyr                 1540  #               1545   #              1550 caa ttc aac gct ctt gag att gat ggt tct gg #c ctc aac ttc atc aac     4704 Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gl #y Leu Asn Phe Ile Asn             1555      #           1560       #          1565 aac tct gct tcc att gat gtc acc ttc act gc #c ttc gct gag gat ggc     4752 Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Al #a Phe Ala Glu Asp Gly         1570          #       1575           #      1580 cgc aag ttg ggt tac gag agc ttc tcc atc cc #a gtc acc ctt aag gtt     4800 Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pr #o Val Thr Leu Lys Val     1585              #   1590               #  1595 tcc act gac aac gca ctc acc ctt cat cac aa #c gag aac ggt gct cag     4848 Ser Thr Asp Asn Ala Leu Thr Leu His His As #n Glu Asn Gly Ala Gln 1600                1605 #                1610  #               1615 tac atg caa tgg caa agc tac cgc acc agg tt #g aac acc ctc ttc gca     4896 Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Le #u Asn Thr Leu Phe Ala                 1620  #               1625   #              1630 agg caa ctt gtg gcc cgt gcc acc aca ggc at #t gac acc atc ctc agc     4944 Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Il #e Asp Thr Ile Leu Ser             1635      #           1640       #          1645 atg gag acc cag aac atc caa gag cca cag tt #g ggc aag ggt ttc tac     4992 Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Le #u Gly Lys Gly Phe Tyr         1650          #       1655           #      1660 gcc acc ttc gtc atc cca cct tac aac ctc ag #c act cat ggt gat gag     5040 Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Se #r Thr His Gly Asp Glu     1665              #   1670               #  1675 agg tgg ttc aag ctc tac atc aag cac gtg gt #t gac aac aac tcc cac     5088 Arg Trp Phe Lys Leu Tyr Ile Lys His Val Va #l Asp Asn Asn Ser His 1680                1685 #                1690  #               1695 atc atc tac tct ggt caa ctc act gac acc aa #c atc aac atc acc ctc     5136 Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr As #n Ile Asn Ile Thr Leu                 1700  #               1705   #              1710 ttc atc cca ctt gac gat gtc cca ctc aac ca #g gac tac cat gcc aag     5184 Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gl #n Asp Tyr His Ala Lys             1715      #           1720       #          1725 gtc tac atg acc ttc aag aag tct cca tct ga #t ggc acc tgg tgg ggt     5232 Val Tyr Met Thr Phe Lys Lys Ser Pro Ser As #p Gly Thr Trp Trp Gly         1730          #       1735           #      1740 cca cac ttc gtc cgt gat gac aag ggc atc gt #c acc atc aac cca aag     5280 Pro His Phe Val Arg Asp Asp Lys Gly Ile Va #l Thr Ile Asn Pro Lys     1745              #   1750               #  1755 tcc atc ctc acc cac ttc gag tct gtc aac gt #t ctc aac aac atc tcc     5328 Ser Ile Leu Thr His Phe Glu Ser Val Asn Va #l Leu Asn Asn Ile Ser 1760                1765 #                1770  #               1775 tct gag cca atg gac ttc tct ggt gcc aac tc #c ctc tac ttc tgg gag     5376 Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Se #r Leu Tyr Phe Trp Glu                 1780  #               1785   #              1790 ttg ttc tac tac aca cca atg ctt gtg gct ca #a agg ttg ctc cat gag     5424 Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gl #n Arg Leu Leu His Glu             1795      #           1800       #          1805 cag aac ttc gat gag gcc aac agg tgg ctc aa #g tac gtc tgg agc cca     5472 Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Ly #s Tyr Val Trp Ser Pro         1810          #       1815           #      1820 tct ggt tac att gtg cat ggt caa atc cag aa #c tac caa tgg aac gtc     5520 Ser Gly Tyr Ile Val His Gly Gln Ile Gln As #n Tyr Gln Trp Asn Val     1825              #   1830               #  1835 agg cca ttg ctt gag gac acc tcc tgg aac tc #t gac cca ctt gac tct     5568 Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Se #r Asp Pro Leu Asp Ser 1840                1845 #                1850  #               1855 gtg gac cct gat gct gtg gct caa cat gac cc #a atg cac tac aag gtc     5616 Val Asp Pro Asp Ala Val Ala Gln His Asp Pr #o Met His Tyr Lys Val                 1860  #               1865   #              1870 tcc acc ttc atg agg acc ttg gac ctc ttg at #t gcc aga ggt gac cat     5664 Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Il #e Ala Arg Gly Asp His             1875      #           1880       #          1885 gct tac cgc caa ttg gag agg gac acc ctc aa #c gag gca aag atg tgg     5712 Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu As #n Glu Ala Lys Met Trp         1890          #       1895           #      1900 tac atg caa gct ctc cac ctc ttg ggt gac aa #g cca tac ctc cca ctc     5760 Tyr Met Gln Ala Leu His Leu Leu Gly Asp Ly #s Pro Tyr Leu Pro Leu     1905              #   1910               #  1915 agc acc act tgg tcc gac cca agg ttg gac cg #t gct gct gac atc acc     5808 Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Ar #g Ala Ala Asp Ile Thr 1920                1925 #                1930  #               1935 act cag aac gct cat gac tct gcc att gtt gc #t ctc agg cag aac atc     5856 Thr Gln Asn Ala His Asp Ser Ala Ile Val Al #a Leu Arg Gln Asn Ile                 1940  #               1945   #              1950 cca act cct gct cca ctc tcc ctc aga tct gc #t aac acc ctc act gac     5904 Pro Thr Pro Ala Pro Leu Ser Leu Arg Ser Al #a Asn Thr Leu Thr Asp             1955      #           1960       #          1965 ttg ttc ctc cca cag atc aac gag gtc atg at #g aac tac tgg caa acc     5952 Leu Phe Leu Pro Gln Ile Asn Glu Val Met Me #t Asn Tyr Trp Gln Thr         1970          #       1975           #      1980 ttg gct caa agg gtc tac aac ctc aga cac aa #c ctc tcc att gat ggt     6000 Leu Ala Gln Arg Val Tyr Asn Leu Arg His As #n Leu Ser Ile Asp Gly     1985              #   1990               #  1995 caa cca ctc tac ctc cca atc tac gcc aca cc #a gct gac cca aag gct     6048 Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pr #o Ala Asp Pro Lys Ala 2000                2005 #                2010  #               2015 ctt ctc tct gct gct gtg gct acc agc caa gg #t ggt ggc aag ctc cca     6096 Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gl #y Gly Gly Lys Leu Pro                 2020  #               2025   #              2030 gag tcc ttc atg tcc ctc tgg agg ttc cca ca #c atg ttg gag aac gcc     6144 Glu Ser Phe Met Ser Leu Trp Arg Phe Pro Hi #s Met Leu Glu Asn Ala             2035      #           2040       #          2045 cgt ggc atg gtc tcc caa ctc acc cag ttc gg #t tcc acc ctc cag aac     6192 Arg Gly Met Val Ser Gln Leu Thr Gln Phe Gl #y Ser Thr Leu Gln Asn         2050          #       2055           #      2060 atc att gag agg caa gat gct gag gct ctc aa #c gct ttg ctc cag aac     6240 Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu As #n Ala Leu Leu Gln Asn     2065              #   2070               #  2075 cag gca gct gag ttg atc ctc acc aac ttg tc #c atc caa gac aag acc     6288 Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Se #r Ile Gln Asp Lys Thr 2080                2085 #                2090  #               2095 att gag gag ctt gat gct gag aag aca gtc ct #t gag aag agc aag gct     6336 Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Le #u Glu Lys Ser Lys Ala                 2100  #               2105   #              2110 ggt gcc caa tct cgc ttc gac tcc tac ggc aa #g ctc tac gat gag aac     6384 Gly Ala Gln Ser Arg Phe Asp Ser Tyr Gly Ly #s Leu Tyr Asp Glu Asn             2115      #           2120       #          2125 atc aac gct ggt gag aac cag gcc atg acc ct #c agg gct tcc gca gct     6432 Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Le #u Arg Ala Ser Ala Ala         2130          #       2135           #      2140 ggt ctc acc act gct gtc caa gcc tct cgc tt #g gct ggt gca gct gct     6480 Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Le #u Ala Gly Ala Ala Ala     2145              #   2150               #  2155 gac ctc gtt cca aac atc ttc ggt ttc gct gg #t ggt ggc tcc aga tgg     6528 Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gl #y Gly Gly Ser Arg Trp 2160                2165 #                2170  #               2175 ggt gcc att gct gag gct acc ggt tac gtc at #g gag ttc tct gcc aac     6576 Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Me #t Glu Phe Ser Ala Asn                 2180  #               2185   #              2190 gtc atg aac act gag gct gac aag atc agc ca #a tct gag acc tac aga     6624 Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gl #n Ser Glu Thr Tyr Arg             2195      #           2200       #          2205 agg cgc cgt caa gag tgg gag atc caa agg aa #c aac gct gag gca gag     6672 Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg As #n Asn Ala Glu Ala Glu         2210          #       2215           #      2220 ttg aag caa atc gat gct caa ctc aag tcc tt #g gct gtc aga agg gag     6720 Leu Lys Gln Ile Asp Ala Gln Leu Lys Ser Le #u Ala Val Arg Arg Glu     2225              #   2230               #  2235 gct gct gtc ctc cag aag acc tcc ctc aag ac #c caa cag gag caa acc     6768 Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Th #r Gln Gln Glu Gln Thr 2240                2245 #                2250  #               2255 cag tcc cag ttg gct ttc ctc caa agg aag tt #c tcc aac cag gct ctc     6816 Gln Ser Gln Leu Ala Phe Leu Gln Arg Lys Ph #e Ser Asn Gln Ala Leu                 2260  #               2265   #              2270 tac aac tgg ctc aga ggc cgc ttg gct gcc at #c tac ttc caa ttc tac     6864 Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Il #e Tyr Phe Gln Phe Tyr             2275      #           2280       #          2285 gac ctt gct gtg gcc agg tgc ctc atg gct ga #g caa gcc tac cgc tgg     6912 Asp Leu Ala Val Ala Arg Cys Leu Met Ala Gl #u Gln Ala Tyr Arg Trp         2290          #       2295           #      2300 gag ttg aac gat gac tcc gcc agg ttc atc aa #g cca ggt gct tgg caa     6960 Glu Leu Asn Asp Asp Ser Ala Arg Phe Ile Ly #s Pro Gly Ala Trp Gln     2305              #   2310               #  2315 ggc acc tac gct ggt ctc ctt gct ggt gag ac #c ctc atg ctc tcc ttg     7008 Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Th #r Leu Met Leu Ser Leu 2320                2325 #                2330  #               2335 gct caa atg gag gat gct cac ctc aag agg ga #c aag agg gct ttg gag     7056 Ala Gln Met Glu Asp Ala His Leu Lys Arg As #p Lys Arg Ala Leu Glu                 2340  #               2345   #              2350 gtg gag agg aca gtc tcc ctt gct gag gtc ta #c gct ggt ctc cca aag     7104 Val Glu Arg Thr Val Ser Leu Ala Glu Val Ty #r Ala Gly Leu Pro Lys             2355      #           2360       #          2365 gac aac ggt cca ttc tcc ctt gct caa gag at #t gac aag ttg gtc agc     7152 Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Il #e Asp Lys Leu Val Ser         2370          #       2375           #      2380 caa ggt tct ggt tct gct ggt tct ggt aac aa #c aac ttg gct ttc ggc     7200 Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn As #n Asn Leu Ala Phe Gly     2385              #   2390               #  2395 gct ggt act gac acc aag acc tcc ctc caa gc #c tct gtc tcc ttc gct     7248 Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Al #a Ser Val Ser Phe Ala 2400                2405 #                2410  #               2415 gac ctc aag atc agg gag gac tac cca gct tc #c ctt ggc aag atc agg     7296 Asp Leu Lys Ile Arg Glu Asp Tyr Pro Ala Se #r Leu Gly Lys Ile Arg                 2420  #               2425   #              2430 cgc atc aag caa atc tct gtc acc ctc cca gc #t ctc ttg ggt cca tac     7344 Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Al #a Leu Leu Gly Pro Tyr             2435      #           2440       #          2445 caa gat gtc caa gca atc ctc tcc tac ggt ga #c aag gct ggt ttg gcg     7392 Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly As #p Lys Ala Gly Leu Ala         2450          #       2455           #      2460 aac ggt tgc gag gct ctt gct gtc tct cat gg #c atg aac gac tct ggt     7440 Asn Gly Cys Glu Ala Leu Ala Val Ser His Gl #y Met Asn Asp Ser Gly     2465              #   2470               #  2475 caa ttc caa ctt gac ttc aac gat ggc aag tt #c ctc cca ttc gag ggc     7488 Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Ph #e Leu Pro Phe Glu Gly 2480                2485 #                2490  #               2495 att gcc att gac caa ggc acc ctc acc ctc tc #c ttc cca aac gct tcc     7536 Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Se #r Phe Pro Asn Ala Ser                 2500  #               2505   #              2510 atg cca gag aag gga aag caa gcc acc atg ct #c aag acc ctc aac gat     7584 Met Pro Glu Lys Gly Lys Gln Ala Thr Met Le #u Lys Thr Leu Asn Asp             2515      #           2520       #          2525 atc atc ctc cac atc agg tac acc atc aag tg #agctc                  #    7621 Ile Ile Leu His Ile Arg Tyr Thr Ile Lys         2530          #       2535 

We claim:
 1. An isolated nucleic acid comprising the sequence of SEQ ID NO:
 4. 2. A transgenic monocot plant cell comprising a nucleic acid comprising the sequence of SEQ ID NO:
 4. 3. A transgenic dicot plant cell comprising a nucleic acid comprising the sequence of SEQ ID NO:
 4. 4. A transgenic plant comprising a nucleic acid of SEQ ID NO:4 that imparts insect resistance.
 5. The transgenic plant of claim 4 wherein the plant is rice.
 6. The transgenic plant of claim 4 wherein the plant is maize.
 7. The transgenic plant of claim 4 wherein the plant is tobacco. 